Anti-TIM-3 antibody

ABSTRACT

Disclosed are an anti-human TIM-3 antibody having high ADCC activity or antibody fragment thereof by screening a monoclonal antibody or antibody fragment thereof which binds to the amino acid sequence of the extracellular region of TIM-3 or its three-dimensional structure and exhibits ADCC activity; a hybridoma which produces the antibody; a DNA encoding the antibody; a vector comprising the DNA; a transformant which is obtainable by introducing the vector; a method for producing the antibody or the antibody fragment thereof which comprises using the hybridoma or the transformant; and a therapeutic agent and a diagnostic agent comprising the antibody or the antibody fragment thereof as an active ingredient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/158,622 issued as U.S. Pat. No. 8,552,156), filed on Jun. 13, 2011,which claims the benefit of U.S. Provisional Application No. 61/353,836,filed on Jun. 11, 2010, the disclosures of which are incorporated hereinin their entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a monoclonal antibody or an antibodyfragment thereof, which binds to the extracellular region of T-cellimmunoglobulin and mucin domain containing molecule-3 (hereinafterreferred to as “TIM-3”) and exhibits antibody-dependent cellularcytotoxicity (hereinafter referred to as “ADCC”); a hybridoma whichproduces the antibody; a DNA which encodes the antibody; a vector whichcomprises the DNA; a transformant obtained by transforming the vector; aprocess for producing an antibody or an antibody fragment thereof usingthe hybridoma or the transformant; and a diagnostic agent or atherapeutic agent using the antibody or the antibody fragment thereof.

2. Brief Description of the Background Art

TIM-3 gene family consists of eight genes in mouse and three genes inhuman, and each of these genes are located at chromosome 11 and atchromosome 5q33 respectively (Non-patent Document 1). These gene regionsare known to be related with autoimmune diseases and allergic diseases.TIM protein is a type I transmembrane protein having a structurallyconserved immunoglobulin variable (IgV) domain and a mucin domain.

TIM protein was considered to be specifically expressed on T cells anddirectly regulate the T cell activity, but there are recent reports onexpression of TIM-3 protein in antigen-presenting cells and on theirfunctions (Non-patent Document 2). According to the crystal structureanalysis, the TIM protein has a conserved protein structure and has aligand binding site in the IgV domain.

TIM-3 was identified as a molecule specifically expressed on mouse Th1cells but not on Th2 cells (Non-patent Document 3). The DNA sequence,the amino acid sequence and the three-dimensional structure of TIM-3 isavailable in the public data base such as the GenBank accession numberNM_032782 and NM_134250. TIM-3 is also known as HAVCR2.

In humans, as similar to mice, TIM-3 is expressed on T-cells as well asphagocytic cells such as macrophages and dendritic cells. Binding ofTIM-3 to a protein ligand (e.g., galectin-9) can inhibit the Th1response via mechanism of apoptosis induction, and therefore lead tosuch as induction of peripheral tolerance.

The reduction in expression of human TIM-3 with siRNA or the inhibitionof human TIM-3 by blocking-antibody increased the secretion ofinterferon γ (IFN-γ) from CD4 positive T-cells, supporting theinhibitory role of TIM-3 in human T cells. In phagocytes, TIM-3 alsofunctions as a receptor for recognizing the apoptosis cells.

Analysis of clinical samples from autoimmune disease patients showed noexpression of TIM-3 in CD4 positive cells. In particular, in T cellclones derived from the cerebrospinal fluid of patients with multiplesclerosis, the expression level of TIM-3 was lower and the secretionlevel of IFN-γ was higher than those of clones derived from normalhealthy persons (Non-patent Document 4). There are reports on relationof TIM-3 with allergic diseases or asthma (Patent Documents 1 and 2).

According to the microarray analysis of hematopoietic stem cells fromacute myeloid leukemia (hereinafter referred to as “AML”) patients andnormal hematopoietic stem cells, TIM-3 is expressed on AML stem cellsand therefore the analysis suggested involvement of TIM-3 inhematological malignancy (Non-patent Document 5 and Patent Document 3).

Examples of the anti-TIM-3 monoclonal antibodies which were establishedup to now include anti-human TIM-3 rat monoclonal antibody (Clone344823, manufactured by R&D Systems) and anti-human TIM-3 mousemonoclonal antibody (Clone F38-2E2, manufactured by R&D Systems).

CITATION LIST Patent Literature

-   [Patent Literature 1] WO96/27603-   [Patent Literature 2] WO2003/063792-   [Patent Literature 3] WO2009/091547

Non-Patent Literature

-   [Non-Patent Literature 1] Hafler D A et al., J Exp Med. 205:    2699-701 (2008)-   [Non-Patent Literature 2] Anderson A C et al., Science 318: 1141-3    (2007)-   [Non-Patent Literature 3] Monney L et al., Nature 415: 536-41 (2002)-   [Non-Patent Literature 4] Koguchi K et al., J Exp Med. 203: 1413-8    (2006)-   [Non-Patent Literature 5] Majeti R et al., Proc Natl Acad Sci USA    2009 Mar. 3; 106 (9): 3396-401.

SUMMARY OF THE INVENTION

However, there is no report on monoclonal antibody against human TIM-3having ADCC activity. Therefore, the object of the present invention isto provide a monoclonal antibody or an antibody fragment which binds tothe amino acid sequence or the three-dimensional structure of theextracellular region of TIM-3 and expresses ADCC activity. In addition,the object of the present invention is to provide an anti-human TIM-3antibody having high ADCC activity by screening an anti-human TIM-3antibody which competes with the monoclonal antibody or the antibodyfragment thereof.

In addition, the present invention is to provide a hybridoma whichproduces the antibody; a DNA which encodes the antibody; a vector whichcomprises the DNA; a transformant obtained by transforming the vector; aprocess for producing an antibody or an antibody fragment thereof usingthe hybridoma or the transformant; and a diagnostic agent or atherapeutic agent using the antibody or the antibody fragment thereof asan active ingredient.

The monoclonal antibody or the antibody fragment of the presentinvention specifically recognizes the amino acid sequence or thethree-dimensional structure of the extracellular region of human TIM-3,and binds to the extracellular region. The amino acid sequence or thethree-dimensional structure of the extracellular region of human TIM-3which is recognized by the monoclonal antibody or the antibody fragmentof the present invention is different from those recognized by knownanti-TIM-3 monoclonal antibodies. Therefore, the monoclonal antibody orthe antibody fragment of the present invention has higher ADCC activity.The monoclonal antibody or the antibody fragment of the presentinvention which specifically binds to the extracellular region of humanTIM-3 and has higher ADCC activity is useful as a therapeutic agent anda diagnostic agent for a disease relating to a human TIM-3 positivecell.

The present invention may provide a hybridoma which produces theantibody; a DNA which encodes the antibody; a vector which comprises theDNA; a transformant obtained by transforming the vector; a process forproducing an antibody or an antibody fragment thereof using thehybridoma or the transformant; and a diagnostic agent or a therapeuticagent using the antibody or the antibody fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the amino acid sequences which represent HV0, HV3, HV4, HV5,HV6, HV7, HV8, HV10, and HV12 of the designed H chain variable regionsof antibody 8213, respectively.

FIG. 2 is the amino acid sequences which represent LV0, LV2, LV4, LV5,LV6, LV7, and LV9 of the designed L chain variable regions of antibody8213.

DETAILED DESCRIPTION OF THE INVENTION

The gist of the present invention relates to the followings.

(1) A monoclonal antibody or an antibody fragment thereof, which bindsto an extracellular region of human TIM-3 while competing with oneantibody selected from the following (i) to (iii):

(i) an antibody which comprises a heavy chain (hereinafter referred toas “H chain”) of an antibody which comprises complementary determiningregions (hereinafter referred to as “CDR”) 1 to 3 comprising the aminoacid sequences represented by SEQ ID NOs: 1 to 3, respectively, andcomprises a light chain (hereinafter referred to as “L chain”) of anantibody which comprises CDRs 1 to 3 comprising the amino acid sequencesrepresented by SEQ ID NOs: 4 to 6, respectively,

(ii) an antibody which comprises an H chain of an antibody whichcomprises CDRs 1 to 3 comprising the amino acid sequences represented bySEQ ID NOs: 11 to 13, respectively, and comprises an L chain of anantibody which comprises CDRs 1 to 3 comprising the amino acid sequencesrepresented by SEQ ID NOs: 14 to 16, respectively,

(iii) an antibody which comprises an H chain of an antibody whichcomprises CDRs1 to 3 comprising the amino acid sequences represented bySEQ ID NOs: 21 to 23, respectively, and comprises an L chain of anantibody which comprises CDRs1 to 3 comprising the amino acid sequencesrepresented by SEQ ID NOs: 24 to 26, respectively.

(2) The monoclonal antibody or the antibody fragment thereof describedin the above (1), wherein the monoclonal antibody binds to the sameepitope to which one antibody selected from the above (i) to (iii)binds.

(3) A monoclonal antibody or an antibody fragment thereof, which bindsto an extracellular region of human TIM-3 while competing with oneantibody selected from the following (a) and (b):

(a) an antibody which comprises VH of an antibody comprising the aminoacid sequence represented by SEQ ID NO: 8 and comprises VL of anantibody comprising the amino acid sequence represented by SEQ ID NO:10,

(b) an antibody which comprises VH of an antibody comprising the aminoacid sequence represented by SEQ ID NO: 18 and comprises VL of anantibody comprising the amino acid sequence represented by SEQ ID NO:20.

(4) The monoclonal antibody or the antibody fragment thereof describedin the above (3), which binds to the same epitope to which one antibodyselected from the above (a) or (b) binds.

(5) The monoclonal antibody or the antibody fragment thereof describedin any one of the above (1) to (4), which is a recombinant antibody.

(6) The monoclonal antibody or the antibody fragment thereof describedin the above (5), which is a recombinant antibody selected from a humanchimeric antibody, a humanized antibody and a human antibody.

(7) A monoclonal antibody or an antibody fragment thereof, which is oneselected from the following (i) to (iii):

(i) a monoclonal antibody and an antibody fragment thereof whichcomprises an H chain of an antibody which comprises CDRs 1 to 3comprising the amino acid sequences represented by SEQ ID NOs: 1 to 3,respectively, and comprises an L chain of an antibody which comprisesCDRs1 to 3 comprising the amino acid sequences represented by SEQ IDNOs: 4 to 6, respectively,

(ii) a monoclonal antibody and an antibody fragment thereof whichcomprises an H chain of an antibody which comprises CDRs 1 to 3comprising the amino acid sequences represented by SEQ ID NOs: 11 to 13,respectively, and comprises an L chain of an antibody which comprisesCDRs 1 to 3 comprising the amino acid sequences represented by SEQ IDNOs: 14 to 16, respectively,

(iii) a monoclonal antibody and an antibody fragment thereof whichcomprises an H chain of an antibody which comprises CDRs 1 to 3comprising the amino acid sequences represented by SEQ ID NOs: 21 to 23,respectively, and comprises an L chain of an antibody which comprisesCDRs 1 to 3 comprising the amino acid sequences represented by SEQ IDNOs: 24 to 26, respectively.

(8) A monoclonal antibody and an antibody fragment thereof, which is oneselected from the following (a) and (b):

(a) a monoclonal antibody and an antibody fragment thereof whichcomprises VH of an antibody comprising the amino acid sequencerepresented by SEQ ID NO: 8 and comprises VL of an antibody comprisingthe amino acid sequence represented by SEQ ID NO: 10,

(b) a monoclonal antibody and an antibody fragment thereof whichcomprises VH of an antibody comprising the amino acid sequencerepresented by SEQ ID NO: 18 and comprises VL of an antibody comprisingthe amino acid sequence represented by SEQ ID NO: 20.

(9) A monoclonal antibody and an antibody fragment thereof which bindsto the amino acid sequences at positions 67 to 105, the amino acidsequences at positions 67 to 96 or the amino acid sequences at positions67 to 87 in the amino acid sequence of IgV domain of human TIM-3represented by SEQ ID NO: 53.

(10) The antibody fragment described in any one of the above (1) to (9),wherein the antibody fragment is an antibody fragment selected from Fab,Fab′, F(ab′)₂, a single chain antibody (scFv), a dimerized V region(diabody), a disulfide stabilized V region (dsFv) and a peptidecomprising CDR.

(11) A DNA which encodes the monoclonal antibody or the antibodyfragment thereof described in any one of the above (1) to (10).

(12) A recombinant vector which comprises the DNA described in the above(11).

(13) A transformant obtainable by introducing the recombinant vectordescribed in the above (12) into a host cell.

(14) A process for producing the monoclonal antibody or the antibodyfragment thereof described in any one of the above (1) to (10), whichcomprises culturing the transformant described in the above (13) inculture to form and accumulate the monoclonal antibody or the antibodyfragment thereof described in any one of the above (1) to (10), andrecovering the monoclonal antibody or the antibody fragment thereof fromthe culture.

(15) A method for immunologically detecting or measuring a human TIM-3which comprises using the monoclonal antibody or the antibody fragmentthereof described in any one of the above (1) to (10).

(16) A reagent for detecting or measuring a human TIM-3 which comprisesthe monoclonal antibody or the antibody fragment thereof described inany one of the above (1) to (10).

(17) A diagnostic agent for a disease relating to a human TIM-3 positivecell which comprises the monoclonal antibody or the antibody fragmentthereof described in any one of the above (1) to (10).

(18) A method for diagnosing a disease relating to a human TIM-3positive cell which comprises detect or measure a human TIM-3 positivecell using the monoclonal antibody or the antibody fragment thereofdescribed in any one of the above (1) to (10).

(19) A method for diagnosing a disease relating to a human TIM-3positive cell, which comprises ditecting or measuring a human TIM-3using the monoclonal antibody or the antibody fragment thereof describedin any one of the above (1) to (10).

(20) Use of the monoclonal antibody or the antibody fragment thereofdescribed in any one of the above (1) to (10) for the manufacture of adiagnostic agent for a disease relating to a human TIM-3 positive cell.

(21) A therapeutic agent for a disease relating to a human TIM-3positive cell, which comprises the monoclonal antibody or the antibodyfragment thereof described in any one of the above (1) to (10).

(22) A therapeutic method for a disease relating to a human TIM-3positive cell, which comprises inducing cell death of a human TIM-3positive cell using the monoclonal antibody or the antibody fragmentthereof described in any one of the above (1) to (10).

(23) Use of the monoclonal antibody or the antibody fragment thereofdescribed in any one of the above (1) to (10) for the manufacture of atherapeutic agent for a disease relating to a human TIM-3 positive cell.

The human TIM-3 of the present invention include a polypeptidecomprising the amino acid sequence represented by SEQ ID NO: 53 or NCBIAccession No. NM_032782, and having a function of TIM-3; a polypeptidecomprising an amino acid sequence having at least 60% homology,preferably at least 80% homology, more preferably at least 90% homology,and most preferably at least 95% homology, with the amino acid sequencerepresented by SEQ ID NO: 53 or NCBI Accession No. NM_032782, and havinga function of TIM-3; and the like.

The polypeptide comprising an amino acid sequence wherein one or moreamino acid residue(s) is/are deleted, substituted and/or added in theamino acid sequence represented by SEQ ID NO: 53 or NCBI Accession No.NM_032782 can be obtained, for example, by introducing a site-directedmutation into DNA encoding the polypeptide comprising the amino acidsequence represented by SEQ ID NO: 53 by site-directed mutagenesisdescribed in Molecular Cloning, A Laboratory Manual, Second Edition(Cold Spring Harbor Laboratory Press, 1989), Current Protocols inMolecular Biology (John Wiley & Sons, 1987-1997), Nucleic AcidsResearch, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982),Gene, 34, 315 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), or thelike.

The number of amino acid residues which are deleted, substituted oradded is not particularly limited, and the number is preferably, 1 todozens, such as 1 to 20, and more preferably 1 to several, such as 1 to5.

As a gene which encodes human TIM-3, examples include the nucleotidesequence represented by SEQ ID NO: 52 or NCBI Accession No. NM_032782.Examples also include a gene comprising a nucleotide sequence in whichat least one nucleotide is deleted, substituted or added in thenucleotide sequence represented by SEQ ID NO: 52 or NCBI Accession No.NM_032782 and comprising a DNA encoding a polypeptide having a functionof TIM-3; a gene comprising an nucleotide sequence having at least 60%homology, preferably at least 80% homology, and more preferably at least95% homology, with the nucleotide sequence represented by SEQ ID NO: 52or NCBI Accession No. NM_032782 and comprising a DNA encoding apolypeptide having a function of TIM-3; a gene which hybridizes with theDNA consisting of the nucleotide sequence represented by SEQ ID NO: 52or NCBI Accession No. NM_032782 under stringent conditions and encodes apolypeptide having a function of TIM-3; and the like.

In the present invention, the DNA which hybridizes under stringentconditions refers to a DNA which is obtained by colony hybridization,plaque hybridization, Southern blot hybridization, DNA microarrayanalysis, or the like using a DNA consisting of the nucleotide sequencerepresented by SEQ ID NO: 52 or NCBI Accession No. NM_032782 as a probe.

A specific example of such DNA is a DNA which can be identified byperforming hybridization at 65° C. in the presence of 0.7 to 1.0 mol/Lsodium chloride using a filter or a slide glass with colony- orplaque-derived DNA, PCR products or oligo DNA encoding the DNA sequenceimmobilized thereon, and then washing the filter or a slide glass at 65°C. with a 0.1 to 2-fold concentration SSC solution (1-fold concentrationSSC solution: 150 mmol/L sodium chloride and 15 mmol/L sodium citrate).Hybridization can be carried out according to the methods described inMolecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989), Current Protocols in Molecular Biology,John Wiley & Sons (1987-1997); DNA Cloning 1: Core Techniques, APractical Approach, Second Edition, Oxford University (1995); and thelike.

Specifically, the DNA capable of hybridization includes DNA having atleast 60% or more homology, preferably 80% or more homology, and morepreferably 95% or more homology to the nucleotide sequence representedby SEQ ID NO: 52 or NCBI Accession No. NM_032782.

In the nucleotide sequence of the gene encoding a protein of aeukaryote, genetic polymorphism is often recognized. The TIM-3 gene usedin the present invention also includes a gene in which smallmodification is generated in the nucleotide sequence by suchpolymorphism.

The number of the homology described in the present invention may be anumber calculated by using a homology search program known by theskilled person, unless otherwise indicated. Regarding the nucleotidesequence, the number may be calculated by using a default parameter inBLAST [J. Mol. Biol., 215, 403 (1990)] or the like, and regarding theamino acid sequence, the number may be calculated by using a defaultparameter in BLAST2 [Nucleic Acids Res., 25, 3389 (1997); Genome Res.,7, 649 (1997);http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html] orthe like.

As the default parameter, G (cost to open gap) is 5 for the nucleotidesequence and 11 for the amino acid sequence; −E (cost to extend gap) is2 for the nucleotide sequence and 1 for the amino acid sequence; −q(penalty for nucleotide mismatch) is −3; −r (reward for nucleotidematch) is 1; −e (expect value) is 10; −W (wordsize) is 11 residues forthe nucleotide sequence and 3 residues for the amino acid sequence; −y(Dropoff (X) for blast extensions in bits) is 20 for blastn and 7 for aprogram other than blastn; −X (X dropoff value for gapped alignment inbits) is 15; and Z (final X dropoff value for gapped alignment in bits)is 50 for blastn and 25 for a program other than blastn(http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html).

The polypeptide comprising a partial sequence of the amino acid sequencerepresented by SEQ ID NO: 53 or NCBI Accession No. NM_032782 can beprepared according to a method known by the skilled person. For example,it can be prepared by deleting a part of DNA encoding the amino acidsequence represented by SEQ ID NO: 52 and culturing a transformant intowhich an expression vector containing the DNA is introduced.

Also, based on the thus prepared polypeptide or DNA, a polypeptidecomprising an amino acid sequence in which one or more amino acid(s)is/are deleted, substituted or added in a partial sequence of the aminoacid sequence represented by SEQ ID NO: 53 or NCBI Accession No.NM_032782 can be prepared in the same manner as described above.

The polypeptide comprising a parcial sequence of the amino acid sequencerepresented by SEQ ID NO: 53 or NCBI Accession No. NM_032782; or thepolypeptide comprising an amino acid sequence in which at least oneamino acid is deleted, substituted or added in a parcial sequence of theamino acid sequence represented by SEQ ID NO: 53 or NCBI Accession No.NM_032782 can also be produced by a chemical synthesis method such asfluorenylmethoxycarbonyl (Fmoc) method or t-butyloxycarbonyl (tBoc)method.

The extracellular region of human TIM-3 of the present inventionincludes, for example, regions predicted by using the amino acidsequence of the polypeptide represented by SEQ ID NO: 53 withconventionally known transmembrane region prediction program SOSUI(http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html), TMHMM ver. 2(http://www.cbs.dtu.dk/services/TMHMM-2.0/), ExPASy Proteomics Server(http://Ca.expasy.org/) or SMART (http://smart.embl-heidelberg.de/).

In the present invention, the amino acid sequence of extracellularregion of human TIM-3 includes the amino acid residues at residues atpositions 1 to 201 in the amino acid sequence represented by SEQ ID NO:53, which is the region of the extracellular region predicted by SMART.

In the present invention, the three-dimensional structure of theextracellular region of human TIM-3 is not limited, so long as theextracellular region of human TIM-3 comprising the amino acid sequencerepresented by SEQ ID NO: 53 or GenBank accession number NM_032782 hasthe same structure as in the natural state. The three-dimensionalstructure of the extracellular region of human TIM-3 in the naturalstate is a natural type of the three-dimensional structure of humanTIM-3 expressed on the surface of the cell membrane.

Regarding the function of human TIM-3, binding of TIM-3 with a proteinligand (e.g., galectin 9) can inhibit the Th1 response thoroughmechanism such as apoptosis induction in Th1 cells, therefore leading toinduction of peripheral tolerance. In phagocytes, human TIM-3 functionsas a receptor for recognizing the apoptosis cells.

Binding of the antibody or antibody fragment thereof in the presentinvention to an amino acid sequence of an extracellular region of humanTIM-3 or a three-dimensional structure thereof can be confirmed by aconventionally known immunological immunological detection method usinghuman TIM-3 expressing cells such as a radioimmuno assay with a solidphase sandwich method or enzyme-linked immunosorbent assay (ELISA), andpreferably a fluorescent cell staining method in which the bindingability of a cell expressing a specified antigen and an antibody for thespecific antigen is confirmed.

Specific examples include a fluorescent antibody staining method usingan FMAT8100HTS System (manufactured by Applied Biosystems) [CancerImmunol. Immunother., 36, 373 (1993)] or the like, a fluorescent cellstaining method using flow cytometry, surface plasmon resonance using aBiacore System (manufactured by GE Healthcare) or the like, and thelike.

Furthermore, a known immunological detection methods [MonoclonalAntibodies—Principles and practice, Third edition, Academic Press(1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory(1988), Monoclonal Antibody Experimental Manual, Kodan-sha Scientific(1987)] and the like can be combined to confirm the binding.

In the present invention, the cell expressing human TIM-3 may be anycell, so long as it expresses human TIM-3, and examples include a cellwhich is naturally present in the human body, a cell line establishedfrom the cell which is naturally present in the human body, a cellobtained by a recombinant technique and the like.

Example of the cell which is naturally present in the human bodyincludes a cell expressing the TIM-3 in the body of a patient sufferingfrom cancer, autoimmune disease or allergic disease, concretely, thecells are Th1 cells, macropharges, dendritic cells, and the like.

Example of a cell line established from the cell which is naturallypresent in the human body include a cell line expressing human TIM-3,among cell lines prepared by establishing the human TIM-3-expressingcells obtained from the above cancer patients, and examples includehuman acute myeloid leukemia cell line KG-1 (ATCC Accession No:CCL-246), human Burkitt's lymphoma cell line Daudi (ATCC Accession No:CCL-213) which are established from a human cell and the like.

Specific examples of the cell obtained by a recombinant technique mayinclude a human TIM-3-expressing cell obtained by introducing anexpression vector comprising a human TIM-3-encoding cDNA into an insectcell, an animal cell or the like, and others.

In addition, the present invention relates to a monoclonal antibodywhich recognizes an amino acid sequence or a three-dimensional structureof an extracellular region of human TIM-3 and exhibits ADCC activity.

The ADCC activity of the present invention is a reaction in which anantibody which is bound to human TIM-3 on the cell surface is bound toFcγRIIIa on the surface of mainly a natural killer cell (hereinafterreferred to as NK cell) through Fc region and therefore a cytotoxicmolecule such as molecules perforin, and granzyme released from an NKcell leads to cell lysis [Clark M, Chemical Immunology, 65, 88 (1997);Gorter A et al., Immunol. Today, 20, 576 (1999)].

The antibody of the present invention may be any antibodies as long asit is an antibody or antibody fragment thereof which recognizes an aminoacid sequence of an extracellular region of human TIM-3 or athree-dimensional structure thereof and binds to the extracellularregion; or an antibody or antibody fragment thereof which binds anextracellular region of human TIM-3 or a three-dimensional structurethereof and have ADCC activity.

Specific example of the antibody of the present invention may include anantibody which binds to the amino acid sequence selected from sequencespreferably at positions 67 to 105, more preferably at positions 67 to96, and most preferably at positions 67 to 87 among the amino acidsequence of extracellular region of human TIM-3 comprising of theresidues at positions 1 to 201 in the sequence represented by SEQ ID NO:53, or the three-dimensional structure thereof.

The example of the antibody includes the monoclonal antibody and theantibody fragment thereof as described in the following (i) to (iii):

(i) a monoclonal antibody or an antibody fragment thereof whichcomprises a heavy chain (hereinafter referred to as “H chain”) of anantibody which comprises complementary determining regions (hereinafterreferred to as “CDR”) 1 to 3 comprising the amino acid sequencesrepresented by SEQ ID NOs: 1 to 3, respectively, and comprises a lightchain (hereinafter referred to as “L chain”) of an antibody whichcomprises CDRs 1 to 3 comprising the amino acid sequences represented bySEQ ID NOs: 4 to 6, respectively.

(ii) a monoclonal antibody or an antibody fragment thereof whichcomprises an H chain of an antibody which comprises CDRs 1 to 3comprising the amino acid sequences represented by SEQ ID NOs: 11 to 13,respectively, and comprises an L chain of an antibody which comprisesCDRs 1 to 3 comprising the amino acid sequences represented by SEQ IDNOs: 14 to 16, respectively.

(iii) a monoclonal antibody or an antibody fragment thereof whichcomprises an H chain of an antibody which comprises CDRs 1 to 3comprising the amino acid sequences represented by SEQ ID NOs: 21 to 23,respectively, and comprises an L chain of an antibody which comprisesCDRs 1 to 3 comprising the amino acid sequences represented by SEQ IDNOs: 24 to 26, respectively.

The example of the monoclonal antibody includes a monoclonal antibodyand an antibody fragment thereof as described in the following (a) and(b):

(a) a monoclonal antibody and an antibody fragment thereof whichcomprises VH of an antibody comprising the amino acid sequencerepresented by SEQ ID NO: 8 and comprises VL of an antibody comprisingthe amino acid sequence represented by SEQ ID NO: 10,

(b) a monoclonal antibody and an antibody fragment thereof whichcomprises VH of an antibody comprising the amino acid sequencerepresented by SEQ ID NO: 18 and comprises VL of an antibody comprisingthe amino acid sequence represented by SEQ ID NO: 20.

In addition, the antibody of the present invention include a monoclonalantibody or an antibody fragment thereof which competes with the abovemonoclonal antibody in the binding of an amino acid sequence of anextracellular region of human TIM-3 or a three-dimensional structurethereof; and a monoclonal antibody or an antibody fragment thereof whichbinds to the same epitope existing on the extracellular region of humanTIM-3 to which the above monoclonal antibody binds.

In the present invention, an antibody which compete with a monoclonalantibody means an antibody which recognizes the same or a part of thesame epitope (also referred to as antigen determinant) on theextracellular region of human TIM-3 as the monoclonal antibody of thepresent invention, and binds to the epitope. The antibody which binds tothe same epitope as the monoclonal antibody of the present inventionmeans an antibody which recognizes and binds to an amino acid sequenceof human TIM-3 which the monoclonal antibody of the present inventionrecognizes.

The monoclonal antibody of the present invention includes an antibodyproduced by a hybridoma and a recombinant antibody produced by atransformant transformed with an expression vector containing a geneencoding an antibody.

The monoclonal antibody is an antibody secreted by a single cloneantibody-producing cell, and recognizes only one epitope (also calledantigen determinant) and has uniform amino acid sequence (primarystructure).

Examples of the epitope include a single amino acid sequence, athree-dimensional structure comprising the amino acid sequence, a sugarchain-bound amino acid sequence, a three-dimensional structurecomprising a sugar chain-bound amino acid sequence, and the like,recognized and bound by a monoclonal antibody.

The monoclonal antibody of the present invention binds preferably to theamino acid residues at positions 22 to 131 of the IgV domain of humanTIM-3 in the extracellular sequence of human TIM-3 represented by SEQ IDNO: 53. Specifically, the amino acid sequence to which the monoclonalantibody of the present invention binds is preferably the amino acidresidues at positions 67 to 105, more preferably the amino acid residuesat positions 67 to 96, and most preferably the amino acid residues atpositions 67 to 87 in the amino acid sequence represented by SEQ ID NO:53.

The epitope to which the monoclonal antibody of the present inventionbinds may preferably be included in the IgV domain of human TIM-3represented by the amino acid residues at positions 22 to 131 in theextracellular region of human TIM-3 represented by SEQ ID NO: 53.Specifically, the epitope to which the monoclonal antibody of thepresent invention binds is preferably included in the amino acidresidues at positions 67 to 105, more preferably included in the aminoacid residues at positions 67 to 96, and most preferably included in theamino acid residues at positions 67 to 87 in the amino acid sequencerepresented by SEQ ID NO: 53.

The amino acid sequence of the epitope to which the monoclonal antibodyof the present invention binds may preferably include at least one aminoacid selected from the amino acid residues at positions 67 to 87 in theamino acid sequence represented by SEQ ID NO: 53 in the IgV domain ofhuman TIM-3, and more preferably include at least one amino acidselected from the amino acids at position 67, position 74, position 76,position 78, position 79, position 81, position 83 and position 85.

The amino acid sequence of the epitope to which the monoclonal antibodyof the present invention binds may preferably include at least two ormore continuous amino acids selected from positions 67 to 87 of thehuman TIM-3 IgV domain; and more preferably may include at least oneamino acid selected from the amino acids at position 67, position 74,position 76, position 78, position 79, position 81, position 83 andposition 85 and include at least two or more continuous amino acidsselected from the amino acid residues at positions 67 to 87 in the aminoacid sequence represented by SEQ ID NO: 53.

Specifically, examples of the amino acid sequence of the epitope towhich the monoclonal antibody of the present invention binds include theamino acid sequence comprising the amino acids at positions 67 to 74,the amino acid sequence comprising the amino acids at positions 67 to76, the amino acid sequence comprising the amino acids at positions 67to 78, the amino acid sequence comprising the amino acids at positions67 to 79, the amino acid sequence comprising the amino acids atpositions 67 to 81, the amino acid sequence comprising the amino acidsat positions 67 to 83, the amino acid sequence comprising the aminoacids at positions 67 to 85,

the amino acid sequence comprising the amino acids at positions 74 to76, the amino acid sequence comprising the amino acids at positions 74to 78, the amino acid sequence comprising the amino acids at positions74 to 79, the amino acid sequence comprising the amino acids atpositions 74 to 81, the amino acid sequence comprising the amino acidsat positions 74 to 83, the amino acid sequence comprising the aminoacids at positions 74 to 85,

the amino acid sequence comprising the amino acids at positions 76 to78, the amino acid sequence comprising the amino acids at positions 76to 79, the amino acid sequence comprising the amino acids at positions76 to 81, the amino acid sequence comprising the amino acids atpositions 76 to 83, the amino acid sequence comprising the amino acidsat positions 76 to 85, the amino acid sequence comprising the aminoacids at positions 78 to 79, the amino acid sequence comprising theamino acids at positions 78 to 81, the amino acid sequence comprisingthe amino acids at positions 78 to 83, the amino acid sequencecomprising the amino acids at positions 78 to 85,

the amino acid sequence comprising the amino acids at positions 79 to81, the amino acid sequence comprising the amino acids at positions 79to 83, the amino acid sequence comprising the amino acids at positions79 to 85,

the amino acid sequence comprising the amino acids at position 81 to 83,the amino acid sequence comprising the amino acids at positions 81 to85,

the amino acid sequence comprising the amino acids at positions 83 to85, and the like,

in the amino acid sequence represented by SEQ ID NO: 53.

The hybridoma can be prepared, for example, by preparing the above cellexpressing TIM-3 as an antigen, inducing an antibody-producing cellhaving antigen specificity from an animal immunized with the antigen,and fusing the antibody-producing cell with a myeloma cell. Theanti-TIM-3 antibody can be obtained by culturing the hybridoma oradministering the hybridoma cell into an animal to cause ascites tumorin the animal and separating and purifying the culture or the ascites.

The animal immunized with an antigen may be any animal, so long as ahybridoma can be prepared, and mouse, rat, hamster, chicken, rabbit orthe like is suitably used. Also, the cell having antibody-producingactivity can be obtained from such an animal, and the antibody of thepresent invention includes an antibody produced by a hybridoma obtainedby fusion of the cell after in vitro immunization with a myeloma cell.

In the present invention, the recombinant antibody includes an antibodyproduced by gene recombination, such as a human chimeric antibody, ahuman CDR grafted antibody, a human antibody and an antibody fragmentthereof. Among the recombinant antibodies, one having character of acommon monoclonal antibody, low immunogenicity and prolonged half-lifein blood is preferable as a therapeutic agent. Examples of therecombinant antibody include an antibody which is prepared by modifyingthe above monoclonal antibody of the present invention using arecombinant technique.

The human chimeric antibody is an antibody comprising VH and VL of anantibody of a non-human animal, and a heavy chain constant region(hereinafter referred to as CH) and a light chain constant region(hereinafter referred to as CL) of a human antibody. Specifically, thehuman chimeric antibody of the present invention can be produced byobtaining cDNAs encoding VH and VL from a hybridoma which produces amonoclonal antibody which specifically recognizes human TIM-3 and bindsto an amino acid sequence of extracellular region or a three-dimensionalstructure thereof, inserting each of them into an expression vector foranimal cell comprising DNAs encoding CH and CL of human antibody tothereby construct a vector for expression of human chimeric antibody,and then introducing the vector into an animal cell to express theantibody.

As the CH of the human chimeric antibody, any CH can be used, so long asit belongs to human immunoglobulin (hereinafter referred to as “hIg”),and those belonging to the hIgG class are preferred, and any one of thesubclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3 andhIgG4, can be used. As the CL of the human chimeric antibody, any CL canbe used, so long as it belongs to the hIg class, and those belonging toκ class or λ class can be used.

Specific examples of the human chimeric antibody of the presentinvention includes a chimeric antibody which comprises VH of theantibody comprising the amino acid sequence represented by SEQ ID NO: 28and comprises VL of the antibody comprising the amino acid sequencerepresented by SEQ ID NO: 30.

In addition, the chimeric antibody of the present invention include achimeric antibody which competes with the above monoclonal antibody inthe binding of an extracellular region of human TIM-3 or athree-dimensional structure thereof; and a chimeric antibody which bindsto the same epitope existing on the extracellular region of human TIM-3to which the above chimeric antibody binds.

The human CDR grafted antibody is also called as a humanized antibodyand is an antibody in which CDR of VH and VL of a non-human animalantibody were grafted into the appropriate site of VH and VL of a humanantibody. The human CDR grafted antibody of the present invention can beproduced by constructing cDNAs encoding an antibody V region in whichthe amino acid sequences of CDRs of VH and VL of an antibody derivedfrom a non-human animal produced by a hybridoma which produces amonoclonal antibody which specifically recognizes three-dimensionalstructure of TIM-3 and binds to an amino acid sequence of theextracellular region or three-dimensional structure thereof are graftedinto frameworks (hereinafter referred to as “FR”) of VH and VL of asuitable human antibody, inserting each of them into an expressionvector for animal cell comprising genes encoding CH and CL of a humanantibody to thereby construct a vector for expression of humanizedantibody, and introducing it into an animal cell to thereby express andproduce the humanized antibody.

As the CH of the human CDR grafted antibody, any CH can be used, so longas it belongs to human immunoglobulin (hereinafter referred to as“hIg”), and those belonging to the hIgG class are preferred, and any oneof the subclasses belonging to the hIgG class, such as hIgG1, hIgG2,hIgG3 and hIgG4, can be used. As the CL of the human CDR graftedantibody, any CL can be used, so long as it belongs to the hIg class,and those belonging to κ class or λ class can be used.

Examples of the CDR grafted antibody of the present invention include ahumanized antibody in which CDRs 1 to 3 of VH of the antibody comprisesthe amino acid sequences represented by SEQ ID NO: 21, 22, and 23,respectively, and CDRs 1 to 3 of VL of the antibody comprises the aminoacid sequences represented by SEQ ID NO: 24, 25, and 26, respectively.

Specific examples of the humanized antibody include a humanized antibodycomprising at least one of the following (a) VH and (b) VL:

(a) VH of the antibody comprising the amino acid sequence represented bySEQ ID NO: 67, or an amino acid sequence in which at least one aminoacid residue selected from Lys at position 12, Val at position 20, Argat position 38, Ala at position 40, Met at position 48, Arg at position67, Val at position 68, Ile at position 70, Ala at position 72, Thr atposition 74, Arg at position 98 and Val at position 113 in the aminoacid sequence represented by SEQ ID NO: 67 is substituted with otheramino acid residue(s),

(b) VL of the antibody comprising the amino acid sequence represented bySEQ ID NO: 69, or an amino acid sequence in which at least one aminoacid residue selected from Leu at position 11, Ala at position 13, Valat position 15, Tyr at position 36, Ala at position 43, Pro at position44, Leu at position 46, Phe at position 71 and Thr at position 85 in theamino acid sequence represented by SEQ ID NO: 69 is substituted withother amino acid residue(s).

As the VH comprised in the humanized antibody, the following (1) to (8)are preferable:

(1) VH comprising an amino acid sequence in which Lys at position 12,Val at position 20, Arg at position 38, Ala at position 40, Met atposition 48, Arg at position 67, Val at position 68, Ile at position 70,Ala at position 72, Thr at position 74, Arg at position 98 and Val atposition 113 in the amino acid sequence represented by SEQ ID NO: 67 aresubstituted with other amino acid residues;

(2) VH comprising an amino acid sequence in which Lys at position 12,Arg at position 38, Ala at position 40, Met at position 48, Arg atposition 67, Val at position 68, Ile at position 70, Ala at position 72,Thr at position 74 and Arg at position 98 in the amino acid sequencerepresented by SEQ ID NO: 67 are substituted with other amino acidresidues;

(3) VH comprising an amino acid sequence in which Val at position 20,Arg at position 38, Met at position 48, Val at position 68, Ile atposition 70, Ala at position 72, Arg at position 98 and Val at position113 in the amino acid sequence represented by SEQ ID NO: 67 aresubstituted with other amino acid residues;

(4) VH comprising an amino acid sequence in which Arg at position 38,Ala at position 40, Met at position 48, Arg at position 67, Ala atposition 72, Thr at position 74 and Arg at position 98 in the amino acidsequence represented by SEQ ID NO: 67 are substituted with other aminoacid residues;

(5) VH comprising an amino acid sequence in which Lys at position 12,Arg at position 67, Val at position 68, Ala at position 72, Thr atposition 74 and Arg at position 98 in the amino acid sequencerepresented by SEQ ID NO: 67 are substituted with other amino acidresidues;

(6) VH comprising an amino acid sequence in which Arg at position 38,Ala at position 40, Met at position 48, Arg at position 67 and Arg atposition 98 in the amino acid sequence represented by SEQ ID NO: 67 aresubstituted with other amino acid residues,

(7) VH comprising an amino acid sequence in which Arg at position 38,Met at position 48, Arg at position 67 and Thr at position 74 in theamino acid sequence represented by SEQ ID NO: 67 are substituted withother amino acid residues; and

(8) VH comprising an amino acid sequence in which Arg at position 38,Met at position 48 and Arg at position 98 in the amino acid sequencerepresented by SEQ ID NO: 67 are substituted with other amino acidresidues.

The amino acid sequence of the above VH obtained by the amino acidmodifications includes an amino acid sequence in which at least onemodification among amino acid modifications for substituting Lys atposition 12 with Val, Val at position 20 with Leu, Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala, Ile at position 70with Leu, Ala at position 72 with Val, Thr at position 74 with Lys, Argat position 98 with Gly or Val at position 113 with Leu is introduced inthe amino acid sequence represented by SEQ ID NO: 67.

Specific examples of the amino acid sequence of VH in which twelvemodifications are introduced include an amino acid sequence in whichsubstitutions of Lys at position 12 with Val, Val at position 20 withLeu, Arg at position 38 with Lys, Ala at position 40 with Arg, Met atposition 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67.

Examples of the amino acid sequence of VH in which eleven modificationsare introduced include the following amino acid sequence (1) to (12):

(1) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Ala at position 40 with Arg, Metat position 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67;

(2) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Ala at position 40 with Arg, Metat position 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67;

(3) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Ala at position 40 with Arg, Metat position 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67;

(4) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Metat position 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67;

(5) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Alaat position 40 with Arg, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67;

(6) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Alaat position 40 with Arg, Met at position 48 with Ile, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67;

(7) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Alaat position 40 with Arg, Met at position 48 with Ile, Arg at position 67with Lys, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys, Arg at position 98 with Gly and

Val at position 113 with Leu are introduced in the amino acid sequencerepresented by SEQ ID NO: 67;

(8) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Alaat position 40 with Arg, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ala at position 72 with Val, Thrat position 74 with Lys, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67;

(9) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Alaat position 40 with Arg, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ile at position 70 with Leu, Thrat position 74 with Lys, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67;

(10) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Alaat position 40 with Arg, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ile at position 70 with Leu, Alaat position 72 with Val, Arg at position 98 with Gly and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67;

(11) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Alaat position 40 with Arg, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ile at position 70 with Leu, Alaat position 72 with Val, Thr at position 74 with Lys and Val at position113 with Leu are introduced in the amino acid sequence represented bySEQ ID NO: 67; and

(12) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Alaat position 40 with Arg, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ile at position 70 with Leu, Alaat position 72 with Val, Thr at position 74 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67.

Specific examples of the amino acid sequence of VH in which tenmodifications are introduced include the following amino acid sequence(1) to (8):

(1) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Ala at position 40 with Arg, Metat position 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(2) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Metat position 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(3) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala, Ile at position 70with Leu, Ala at position 72 with Val, Thr at position 74 with Lys, Argat position 98 with Gly and Val at position 113 with Leu are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(4) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala, Ile at position 70with Leu, Ala at position 72 with Val, Thr at position 74 with Lys, Argat position 98 with Gly and Val at position 113 with Leu are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(5) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Ala at position 40 with Arg, Metat position 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(6) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala, Ile at position 70with Leu, Ala at position 72 with Val, Thr at position 74 with Lys, Argat position 98 with Gly and Val at position 113 with Leu are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(7) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Alaat position 40 with Arg, Met at position 48 with Ile, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Argat position 98 with Gly and Val at position 113 with Leu are introducedin the amino acid sequence represented by SEQ ID NO: 67; and

(8) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Ala at position 40 with Arg, Metat position 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Ala at position 72 with Val, Thrat position 74 with Lys and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67.

Examples of the amino acid sequence of VH in which eight modificationsare introduced include the following amino acid sequence (1) to (13):

(1) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Met at position 48 with Ile, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val, Arg at position 98 with Gly and Val at position 113 with Leuare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(2) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Val at position 68 with Ala, Ileat position 70 with Leu, Ala at position 72 with Val, Thr at position 74with Lys, Arg at position 98 with Gly and Val at position 113 with Leuare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(3) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val, Arg at position 98 with Gly and Val at position 113 with Leuare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(4) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val, Thr at position 74 with Lys and Arg at position 98 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(5) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val, Arg at position 98 with Gly and Val at position 113 with Leuare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(6) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val, Thr at position 74 with Lys and Arg at position 98 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(7) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala, Ile at position 70with Leu, Ala at position 72 with Val and Arg at position 98 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(8) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Met at position 48 with Ile, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val, Thr at position 74 with Lys and Arg at position 98 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(9) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Ala at position 40 with Arg, Metat position 48 with Ile, Val at position 68 with Ala, Ile at position 70with Leu, Ala at position 72 with Val and Arg at position 98 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(10) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Met at position 48 with Ile, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val, Arg at position 98 with Gly and Val at position 113 with Leuare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(11) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Met at position 48 with Ile, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val, Thr at position 74 with Lys and Arg at position 98 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(12) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Ala at position 40 with Arg, Metat position 48 with Ile, Val at position 68 with Ala, Ile at position 70with Leu, Ala at position 72 with Val and Arg at position 98 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 67;and

(13) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Metat position 48 with Ile, Val at position 68 with Ala, Ile at position 70with Leu, Ala at position 72 with Val and Arg at position 98 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 67.

Specific examples of the amino acid sequence of VH in which sevenmodifications are introduced include the following amino acid sequence(1) to (11):

(1) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Ala at position 72 with Val, Thr at position 74with Lys and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(2) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Val at position 68 with Ala, Ileat position 70 with Leu, Ala at position 72 with Val, Thr at position 74with Lys and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(3) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Ileat position 70 with Leu, Ala at position 72 with Val, Thr at position 74with Lys and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(4) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Valat position 68 with Ala, Ala at position 72 with Val, Thr at position 74with Lys and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(5) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(6) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Ileat position 70 with Leu, Ala at position 72 with Val, Thr at position 74with Lys and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(7) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Valat position 68 with Ala, Ala at position 72 with Val, Thr at position 74with Lys and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(8) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Valat position 68 with Ala, Ile at position 70 with Leu, Ala at position 72with Val and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(9) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Ala at position 72 with Val, Thr at position 74with Lys and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(10) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Ile at position 70 with Leu, Ala at position 72with Val and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67; and

(11) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala, Ala at position 72with Val and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67.

Specific Examples of the amino acid sequence of VH in which sixmodifications are introduced include the following amino acid sequence(1) to (16):

(1) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 67 with Lys, Val at position 68 with Ala, Alaat position 72 with Val, Thr at position 74 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(2) an amino acid sequence in which substitutions of Lys at position 12with Val, Val at position 20 with Leu, Arg at position 38 with Lys, Metat position 48 with Ile, Arg at position 67 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(3) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Ala at position 40 with Arg, Metat position 48 with Ile, Arg at position 67 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(4) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(5) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys, Ala at position 72 with Val and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(6) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys, Thr at position 74 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(7) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Ala at position 40 with Arg, Metat position 48 with Ile, Arg at position 67 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(8) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(9) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys, Ala at position 72 with Val and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(10) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys, Thr at position 74 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(11) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(12) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Ala at position 72 with Val and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(13) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys, Thr at position 74 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(14) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Valat position 68 with Ala, Ala at position 72 with Val and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67;

(15) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Valat position 68 with Ala, Thr at position 74 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67; and

(16) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Alaat position 72 with Val, Thr at position 74 with Lys and Arg at position98 with Gly are introduced in the amino acid sequence represented by SEQID NO: 67.

Specific examples of the amino acid sequence of VH in which fivemodifications are introduced include the following amino acid sequence(1) to (7):

(1) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile, Argat position 67 with Lys and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(2) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(3) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Met at position 48 with Ile, Argat position 67 with Lys and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(4) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Valat position 68 with Ala and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(5) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Ileat position 70 with Leu and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67;

(6) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Alaat position 72 with Val and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67; and

(7) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys, Thrat position 74 with Lys and Arg at position 98 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 67.

Specific examples of the amino acid sequence of VH in which fourmodifications are introduced include the following amino acid sequence(1) to (8):

(1) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys andThr at position 74 with Lys are introduced in the amino acid sequencerepresented by SEQ ID NO: 67;

(2) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys, Met at position 48 with Ile andArg at position 67 with Lys are introduced in the amino acid sequencerepresented by SEQ ID NO: 67;

(3) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys, Met at position 48 with Ile andArg at position 67 with Lys are introduced in the amino acid sequencerepresented by SEQ ID NO: 67;

(4) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg, Met at position 48 with Ile andArg at position 67 with Lys are introduced in the amino acid sequencerepresented by SEQ ID NO: 67;

(5) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys andVal at position 68 with Ala are introduced in the amino acid sequencerepresented by SEQ ID NO: 67;

(6) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys andIle at position 70 with Leu are introduced in the amino acid sequencerepresented by SEQ ID NO: 67;

(7) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys andAla at position 72 with Val are introduced in the amino acid sequencerepresented by SEQ ID NO: 67; and

(8) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile, Arg at position 67 with Lys andArg at position 98 with Gly are introduced in the amino acid sequencerepresented by SEQ ID NO: 67.

Specific examples of the amino acid sequence of VH in which threemodifications are introduced include the following amino acid sequence(1) to (9):

(1) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile and Arg at position 98 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(2) an amino acid sequence in which substitutions of Lys at position 12with Val, Arg at position 38 with Lys and Met at position 48 with Ileare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(3) an amino acid sequence in which substitutions of Val at position 20with Leu, Arg at position 38 with Lys and Met at position 48 with Ileare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(4) an amino acid sequence in which substitutions of Arg at position 38with Lys, Ala at position 40 with Arg and Met at position 48 with Ileare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(5) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile and Arg at position 67 with Lysare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(6) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile and Val at position 68 with Alaare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(7) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile and Ile at position 70 with Leuare introduced in the amino acid sequence represented by SEQ ID NO: 67;

(8) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile and Ala at position 72 with Valare introduced in the amino acid sequence represented by SEQ ID NO: 67;and

(9) an amino acid sequence in which substitutions of Arg at position 38with Lys, Met at position 48 with Ile and Thr at position 74 with Lysare introduced in the amino acid sequence represented by SEQ ID NO: 67;

Specific examples of the amino acid sequence of VH in which twomodifications are introduced include the following amino acid sequence(1) to (21):

(1) an amino acid sequence in which substitutions of Lys at position 12with Val and Arg at position 38 with Lys are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(2) an amino acid sequence in which substitutions of Val at position 20with Leu and Arg at position 38 with Lys are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(3) an amino acid sequence in which substitutions of Arg at position 38with Lys and Ala at position 40 with Arg are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(4) an amino acid sequence in which substitutions of Arg at position 38with Lys and Met at position 48 with Ile are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(5) an amino acid sequence in which substitutions of Arg at position 38with Lys and Arg at position 67 with Lys are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(6) an amino acid sequence in which substitutions of Arg at position 38with Lys and Val at position 68 with Ala are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(7) an amino acid sequence in which substitutions of Arg at position 38with Lys and Ile at position 70 with Leu are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(8) an amino acid sequence in which substitutions of Arg at position 38with Lys and Ala at position 72 with Val are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(9) an amino acid sequence in which substitutions of Arg at position 38with Lys and Thr at position 74 with Lys are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(10) an amino acid sequence in which substitutions of Arg at position 38with Lys and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(11) an amino acid sequence in which substitutions of Arg at position 38with Lys and Val at position 113 with Leu are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(12) an amino acid sequence in which substitutions of Lys at position 12with Val and Met at position 48 with Ile are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(13) an amino acid sequence in which substitutions of Val at position 20with Leu and Met at position 48 with Ile are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(14) an amino acid sequence in which substitutions of Ala at position 40with Arg and Met at position 48 with Ile are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(15) an amino acid sequence in which substitutions of Met at position 48with Ile and Arg at position 67 with Lys are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(16) an amino acid sequence in which substitutions of Met at position 48with Ile and Val at position 68 with Ala are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(17) an amino acid sequence in which substitutions of Met at position 48with Ile and Ile at position 70 with Leu are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(18) an amino acid sequence in which substitutions of Met at position 48with Ile and Ala at position 72 with Val are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(19) an amino acid sequence in which substitutions of Met at position 48with Ile and Thr at position 74 with Lys are introduced in the aminoacid sequence represented by SEQ ID NO: 67;

(20) an amino acid sequence in which substitutions of Met at position 48with Ile and Arg at position 98 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 67; and

(21) an amino acid sequence in which substitutions of Met at position 48with Ile and Val at position 113 with Leu are introduced in the aminoacid sequence represented by SEQ ID NO: 67.

Specific examples of the amino acid sequence of VH in which onemodification is introduced include the following amino acid sequence (1)to (12):

(1) an amino acid sequence in which substitution of Lys at position 12with Val is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(2) an amino acid sequence in which substitution of Val at position 20with Leu is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(3) an amino acid sequence in which substitution of Arg at position 38with Lys is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(4) an amino acid sequence in which substitution of Ala at position 40with Arg is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(5) an amino acid sequence in which substitution of Met at position 48with Ile is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(6) an amino acid sequence in which substitution of Arg at position 67with Lys is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(7) an amino acid sequence in which substitution of Val at position 68with Ala is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(8) an amino acid sequence in which substitution of Ile at position 70with Leu is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(9) an amino acid sequence in which substitution of Ala at position 72with Val is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(10) an amino acid sequence in which substitution of Thr at position 74with Lys is introduced in the amino acid sequence represented by SEQ IDNO: 67;

(11) an amino acid sequence in which substitution of Arg at position 98with Gly is introduced in the amino acid sequence represented by SEQ IDNO: 67; and

(12) an amino acid sequence in which substitution of Val at position 113with Leu is introduced in the amino acid sequence represented by SEQ IDNO: 67.

As the VL comprised in the humanized antibody, the following (1) to (6)are preferable:

(1) VL comprising an amino acid sequence in which Leu at position 11,Ala at position 13, Val at position 15, Tyr at position 36, Ala atposition 43, Pro at position 44, Leu at position 46, Phe at position 71and Thr at position 85 in the amino acid sequence represented by SEQ IDNO: 69 are substituted with other amino acid residues.

(2) VL comprising an amino acid sequence in which Leu at position 11,Val at position 15, Tyr at position 36, Pro at position 44, Leu atposition 46, Phe at position 71 and Thr at position 85 in the amino acidsequence represented by SEQ ID NO: 69 are substituted with other aminoacid residues.

(3) VL comprising an amino acid sequence in which Val at position 15,Tyr at position 36, Pro at position 44, Leu at position 46, Phe atposition 71 and Thr at position 85 in the amino acid sequencerepresented by SEQ ID NO: 69 are substituted with other amino acidresidues.

(4) VL comprising an amino acid sequence in which Tyr at position 36,Ala at position 43, Pro at position 44, Leu at position 46 and Thr atposition 85 in the amino acid sequence represented by SEQ ID NO: 69 aresubstituted with other amino acid residues.

(5) VL comprising an amino acid sequence in which Val at position 15,Tyr at position 36, Leu at position 46 and Phe at position 71 in theamino acid sequence represented by SEQ ID NO: 69 are substituted withother amino acid residues.

(6) VL comprising an amino acid sequence in which Tyr at position 36 andPro at position 44 in the amino acid sequence represented by SEQ ID NO:69 are substituted with other amino acid residues.

The amino acid sequence of the above VL obtained by the amino acidmodifications includes an amino acid sequence in which at least onemodification among amino acid modifications for substituting Leu atposition 11 with Met, Ala at position 13 with Val, Val at position 15with Leu, Tyr at position 36 with Leu, Ala at position 43 with Ser, Proat position 44 with Phe, Leu at position 46 with Gly, Phe at position 71with Tyr, and Thr at position 85 with Asp is introduced in the aminoacid sequence represented by SEQ ID NO: 69.

Examples of the amino acid sequence of VL in which nine modificationsare introduced include an amino acid sequence in which substitutions ofLeu at position 11 with Met, Ala at position 13 with Val, Val atposition 15 with Leu, Tyr at position 36 with Leu, Ala at position 43with Ser, Pro at position 44 with Phe, Leu at position 46 with Gly, Pheat position 71 with Tyr, and Thr at position 85 with Asp are introducedin the amino acid sequence represented by SEQ ID NO: 69.

Specific examples of the amino acid sequence of VL in which eightmodifications are introduced include the following amino acid sequence(1) to (9):

(1) an amino acid sequence in which substitutions of Ala at position 13with Val, Val at position 15 with Leu, Tyr at position 36 with Leu, Alaat position 43 with Ser, Pro at position 44 with Phe, Leu at position 46with Gly, Phe at position 71 with Tyr and Thr at position 85 with Aspare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(2) an amino acid sequence in which substitutions of Leu at position 11with Met, Val at position 15 with Leu, Tyr at position 36 with Leu, Alaat position 43 with Ser, Pro at position 44 with Phe, Leu at position 46with Gly, Phe at position 71 with Tyr and Thr at position 85 with Aspare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(3) an amino acid sequence in which substitutions of Leu at position 11with Met, Ala at position 13 with Val, Tyr at position 36 with Leu, Alaat position 43 with Ser, Pro at position 44 with Phe, Leu at position 46with Gly, Phe at position 71 with Tyr and Thr at position 85 with Aspare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(4) an amino acid sequence in which substitutions of Leu at position 11with Met, Ala at position 13 with Val, Val at position 15 with Leu, Alaat position 43 with Ser, Pro at position 44 with Phe, Leu at position 46with Gly, Phe at position 71 with Tyr and Thr at position 85 with Aspare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(5) an amino acid sequence in which substitutions of Leu at position 11with Met, Ala at position 13 with Val, Val at position 15 with Leu, Tyrat position 36 with Leu, Pro at position 44 with Phe, Leu at position 46with Gly, Phe at position 71 with Tyr and Thr at position 85 with Aspare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(6) an amino acid sequence in which substitutions of Leu at position 11with Met, Ala at position 13 with Val, Val at position 15 with Leu, Tyrat position 36 with Leu, Ala at position 43 with Ser, Leu at position 46with Gly, Phe at position 71 with Tyr and Thr at position 85 with Aspare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(7) an amino acid sequence in which substitutions of Leu at position 11with Met, Ala at position 13 with Val, Val at position 15 with Leu, Tyrat position 36 with Leu, Ala at position 43 with Ser, Pro at position 44with Phe, Phe at position 71 with Tyr and Thr at position 85 with Aspare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(8) an amino acid sequence in which substitutions of Leu at position 11with Met, Ala at position 13 with Val, Val at position 15 with Leu, Tyrat position 36 with Leu, Ala at position 43 with Ser, Pro at position 44with Phe, Leu at position 46 with Gly and Thr at position 85 with Aspare introduced in the amino acid sequence represented by SEQ ID NO: 69;and

(9) an amino acid sequence in which substitutions of Leu at position 11with Met, Ala at position 13 with Val, Val at position 15 with Leu, Tyrat position 36 with Leu, Ala at position 43 with Ser, Pro at position 44with Phe, Leu at position 46 with Gly and Phe at position 71 with Tyrare introduced in the amino acid sequence represented by SEQ ID NO: 69.

Specific example of the amino acid sequence of VL in which sevenmodifications are introduced include the following amino acid sequence(1) to (9):

(1) an amino acid sequence in which substitutions of Leu at position 11with Met, Val at position 15 with Leu, Tyr at position 36 with Leu, Proat position 44 with Phe, Leu at position 46 with Gly, Phe at position 71with Tyr and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(2) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Ala at position 43 with Ser, Proat position 44 with Phe, Leu at position 46 with Gly, Phe at position 71with Tyr and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(3) an amino acid sequence in which substitutions of Leu at position 11with Met, Tyr at position 36 with Leu, Ala at position 43 with Ser, Proat position 44 with Phe, Leu at position 46 with Gly, Phe at position 71with Tyr and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(4) an amino acid sequence in which substitutions of Leu at position 11with Met, Val at position 15 with Leu, Ala at position 43 with Ser, Proat position 44 with Phe, Leu at position 46 with Gly, Phe at position 71with Tyr and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(5) an amino acid sequence in which substitutions of Leu at position 11with Met, Val at position 15 with Leu, Tyr at position 36 with Leu, Proat position 44 with Phe, Leu at position 46 with Gly, Phe at position 71with Tyr and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(6) an amino acid sequence in which substitutions of Leu at position 11with Met, Val at position 15 with Leu, Tyr at position 36 with Leu, Alaat position 43 with Ser, Leu at position 46 with Gly, Phe at position 71with Tyr and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(7) an amino acid sequence in which substitutions of Leu at position 11with Met, Val at position 15 with Leu, Tyr at position 36 with Leu, Alaat position 43 with Ser, Pro at position 44 with Phe, Phe at position 71with Tyr and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(8) an amino acid sequence in which substitutions of Leu at position 11with Met, Val at position 15 with Leu, Tyr at position 36 with Leu, Alaat position 43 with Ser, Pro at position 44 with Phe, Leu at position 46with Gly and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69; and

(9) an amino acid sequence in which substitutions of Leu at position 11with Met, Val at position 15 with Leu, Tyr at position 36 with Leu, Alaat position 43 with Ser, Pro at position 44 with Phe, Leu at position 46with Gly and Phe at position 71 with Tyr are introduced in the aminoacid sequence represented by SEQ ID NO: 69.

Specific example of the amino acid sequence of VL in which sixmodifications are introduced include the following amino acid sequence(1) to (6):

(1) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Pro at position 44 with Phe, Leuat position 46 with Gly, Phe at position 71 with Tyr and Thr at position85 with Asp are introduced in the amino acid sequence represented by SEQID NO: 69;

(2) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Ala at position 43 with Ser, Pro at position 44 with Phe, Leuat position 46 with Gly, Phe at position 71 with Tyr and Thr at position85 with Asp are introduced in the amino acid sequence represented by SEQID NO: 69;

(3) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Ala at position 43 with Ser, Proat position 44 with Phe, Leu at position 46 with Gly and Thr at position85 with Asp are introduced in the amino acid sequence represented by SEQID NO: 69;

(4) an amino acid sequence in which substitutions of Leu at position 11with Met, Tyr at position 36 with Leu, Pro at position 44 with Phe, Leuat position 46 with Gly, Phe at position 71 with Tyr and Thr at position85 with Asp are introduced in the amino acid sequence represented by SEQID NO: 69;

(5) an amino acid sequence in which substitutions of Leu at position 11with Met, Tyr at position 36 with Leu, Ala at position 43 with Ser, Proat position 44 with Phe, Leu at position 46 with Gly and Thr at position85 with Asp are introduced in the amino acid sequence represented by SEQID NO: 69; and

(6) an amino acid sequence in which substitutions of Leu at position 11with Met, Ala at position 13 with Val, Tyr at position 36 with Leu, Proat position 44 with Phe, Leu at position 46 with Gly and Thr at position85 with Asp are introduced in the amino acid sequence represented by SEQID NO: 69.

Specific example of the amino acid sequence of VL in which fivemodifications are introduced include the following amino acid sequence(1) to (7):

(1) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Ala at position 43 with Ser, Pro at position 44 with Phe, Leuat position 46 with Gly and Thr at position 85 with Asp are introducedin the amino acid sequence represented by SEQ ID NO: 69;

(2) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Ala at position 43 with Ser, Proat position 44 with Phe and Leu at position 46 with Gly are introducedin the amino acid sequence represented by SEQ ID NO: 69;

(3) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Pro at position 44 with Phe, Leuat position 46 with Gly and Phe at position 71 with Tyr are introducedin the amino acid sequence represented by SEQ ID NO: 69;

(4) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Pro at position 44 with Phe, Leuat position 46 with Gly and Thr at position 85 with Asp are introducedin the amino acid sequence represented by SEQ ID NO: 69;

(5) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Ala at position 43 with Ser, Pro at position 44 with Phe, Leuat position 46 with Gly and Phe at position 71 with Tyr are introducedin the amino acid sequence represented by SEQ ID NO: 69;

(6) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Ala at position 43 with Ser, Pro at position 44 with Phe, Leuat position 46 with Gly and Thr at position 85 with Asp are introducedin the amino acid sequence represented by SEQ ID NO: 69; and

(7) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Pro at position 44 with Phe, Leu at position 46 with Gly, Pheat position 71 with Tyr and Thr at position 85 with Asp are introducedin the amino acid sequence represented by SEQ ID NO: 69.

Specific example of the amino acid sequence of VL in which fourmodifications are introduced include the following amino acid sequence(1) to (10):

(1) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Leu at position 46 with Gly andPhe at position 71 with Tyr are introduced in the amino acid sequencerepresented by SEQ ID NO: 69;

(2) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Pro at position 44 with Phe andLeu at position 46 with Gly are introduced in the amino acid sequencerepresented by SEQ ID NO: 69;

(3) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Pro at position 44 with Phe andPhe at position 71 with Tyr are introduced in the amino acid sequencerepresented by SEQ ID NO: 69;

(4) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Pro at position 44 with Phe andThr at position 85 with Asp are introduced in the amino acid sequencerepresented by SEQ ID NO: 69;

(5) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Leu at position 46 with Gly andThr at position 85 with Asp are introduced in the amino acid sequencerepresented by SEQ ID NO: 69;

(6) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu, Phe at position 71 with Tyr andThr at position 85 with Asp are introduced in the amino acid sequencerepresented by SEQ ID NO: 69;

(7) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Pro at position 44 with Phe, Leu at position 46 with Gly andPhe at position 71 with Tyr are introduced in the amino acid sequencerepresented by SEQ ID NO: 69;

(8) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Pro at position 44 with Phe, Leu at position 46 with Gly andThr at position 85 with Asp are introduced in the amino acid sequencerepresented by SEQ ID NO: 69;

(9) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Pro at position 44 with Phe, Phe at position 71 with Tyr andThr at position 85 with Asp are introduced in the amino acid sequencerepresented by SEQ ID NO: 69; and

(10) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Leu at position 46 with Gly, Phe at position 71 with Tyr andThr at position 85 with Asp are introduced in the amino acid sequencerepresented by SEQ ID NO: 69.

Specific example of the amino acid sequence of VL in which threemodifications are introduced include the following amino acid sequence(1) to (7):

(1) an amino acid sequence in which substitutions of Leu at position 11with Met, Tyr at position 36 with Leu and Pro at position 44 with Pheare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(2) an amino acid sequence in which substitutions of Ala at position 13with Val, Tyr at position 36 with Leu and Pro at position 44 with Pheare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(3) an amino acid sequence in which substitutions of Val at position 15with Leu, Tyr at position 36 with Leu and Pro at position 44 with Pheare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(4) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Ala at position 43 with Ser and Pro at position 44 with Pheare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(5) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Pro at position 44 with Phe and Leu at position 46 with Glyare introduced in the amino acid sequence represented by SEQ ID NO: 69;

(6) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Pro at position 44 with Phe and Phe at position 71 with Tyrare introduced in the amino acid sequence represented by SEQ ID NO: 69;and

(7) an amino acid sequence in which substitutions of Tyr at position 36with Leu, Pro at position 44 with Phe and Thr at position 85 with Aspare introduced in the amino acid sequence represented by SEQ ID NO: 69.

Specific example of the amino acid sequence of VL in which twomodifications are introduced include the following amino acid sequence(1) to (15):

(1) an amino acid sequence in which substitutions of Tyr at position 36with Leu and Pro at position 44 with Phe are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(2) an amino acid sequence in which substitutions of Leu at position 11with Met and Tyr at position 36 with Leu are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(3) an amino acid sequence in which substitutions of Ala at position 13with Val and Tyr at position 36 with Leu are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(4) an amino acid sequence in which substitutions of Val at position 15with Leu and Tyr at position 36 with Leu are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(5) an amino acid sequence in which substitutions of Tyr at position 36with Leu and Ala at position 43 with Ser are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(6) an amino acid sequence in which substitutions of Tyr at position 36with Leu and Leu at position 46 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(7) an amino acid sequence in which substitutions of Tyr at position 36with Leu and Phe at position 71 with Tyr are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(8) an amino acid sequence in which substitutions of Tyr at position 36with Leu and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(9) an amino acid sequence in which substitutions of Leu at position 11with Met and Pro at position 44 with Phe are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(10) an amino acid sequence in which substitutions of Ala at position 13with Val and Pro at position 44 with Phe are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(11) an amino acid sequence in which substitutions of Val at position 15with Leu and Pro at position 44 with Phe are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(12) an amino acid sequence in which substitutions of Ala at position 43with Ser and Pro at position 44 with Phe are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(13) an amino acid sequence in which substitutions of Pro at position 44with Phe and Leu at position 46 with Gly are introduced in the aminoacid sequence represented by SEQ ID NO: 69;

(14) an amino acid sequence in which substitutions of Pro at position 44with Phe and Phe at position 71 with Tyr are introduced in the aminoacid sequence represented by SEQ ID NO: 69; and

(15) an amino acid sequence in which substitutions of Pro at position 44with Phe and Thr at position 85 with Asp are introduced in the aminoacid sequence represented by SEQ ID NO: 69.

Specific example of the amino acid sequence of VH in which onemodification is introduced include the following amino acid sequence (1)to (9):

(1) an amino acid sequence in which substitution of Leu at position 11with Met is introduced in the amino acid sequence represented by SEQ IDNO: 69;

(2) an amino acid sequence in which substitution of Ala at position 13with Val is introduced in the amino acid sequence represented by SEQ IDNO: 69;

(3) an amino acid sequence in which substitution of Val at position 15with Leu is introduced in the amino acid sequence represented by SEQ IDNO: 69;

(4) an amino acid sequence in which substitution of Tyr at position 36with Leu is introduced in the amino acid sequence represented by SEQ IDNO: 69;

(5) an amino acid sequence in which substitution of Ala at position 43with Ser is introduced in the amino acid sequence represented by SEQ IDNO: 69;

(6) an amino acid sequence in which substitution of Pro at position 44with Phe is introduced in the amino acid sequence represented by SEQ IDNO: 69;

(7) an amino acid sequence in which substitution of Leu at position 46with Gly is introduced in the amino acid sequence represented by SEQ IDNO: 69;

(8) an amino acid sequence in which substitution of Phe at position 71with Tyr is introduced in the amino acid sequence represented by SEQ IDNO: 69; and

(9) an amino acid sequence in which substitution of Thr at position 85with Asp is introduced in the amino acid sequence represented by SEQ IDNO: 69.

In addition, examples of the humanized antibody of the present inventioninclude a humanized antibody in which VH of the antibody comprises theamino acid sequence represented by SEQ ID NO: 67 and/or VL of theantibody comprises the amino acid sequence represented by SEQ ID: 69, ahumanized antibody in which VH of the antibody comprises the amino acidsequence represented by SEQ ID NO: 67 and/or VL of the antibodycomprises any one of the amino acid sequences shown in FIG. 2, or ahumanized antibody in which VH of the antibody comprises any one of theamino acid sequences shown in FIG. 1 and/or VL of the antibody comprisesthe amino acid sequence represented by SEQ ID: 69.

Furthermore, examples of the humanized antibody of the present inventioninclude a humanized antibody which competes with the monoclonal antibodyof the present invention in the binding of an amino acid sequence of anextracellular region of human TIM-3 or a three-dimensional structurethereof; and a humanized antibody which binds to the same epitopeexisting on the extracellular region of human TIM-3 to which the abovehumanized antibody binds.

A human antibody is originally an antibody naturally existing in thehuman body, and it also includes an antibody obtained from a humanantibody phage library or a human antibody-producing transgenic animal,which is prepared based on the recent advanced techniques in geneticengineering, cell engineering and developmental engineering.

The antibody existing in the human body can be prepared, for example, byisolating a human peripheral blood lymphocyte, immortalizing it byinfecting with EB virus or the like, and then cloning it to therebyobtain lymphocytes capable of producing the antibody, culturing thelymphocytes thus obtained, and purifying the antibody from thesupernatant of the culture.

The human antibody phage library is a library in which antibodyfragments such as Fab and scFv are expressed on the phage surface byinserting a gene encoding an antibody prepared from a human B cell intoa phage gene. A phage expressing an antibody fragment having the desiredantigen binding activity can be recovered from the library, using itsactivity to bind to an antigen-immobilized substrate as the index. Theantibody fragment can be converted further into a human antibodymolecule comprising two full H chains and two full L chains by geneticengineering techniques.

A human antibody-producing transgenic animal is an animal in which ahuman antibody gene is integrated into cells. Specifically, a humanantibody-producing transgenic animal can be prepared by introducing agene encoding a human antibody into a mouse ES cell, grafting the EScell into an early embryo of mouse and then developing it. A humanantibody is prepared from the human antibody-producing transgenicnon-human animal by obtaining a human antibody-producing hybridoma by ahybridoma preparation method usually carried out in non-human mammals,culturing the obtained hybridoma and forming and accumulating the humanantibody in the supernatant of the culture.

In the amino acid sequence constituting the above antibody or antibodyfragment thereof, an antibody or antibody fragment thereof in which oneor more amino acids are deleted, substituted, inserted or added, havingactivity similar to the above antibody or antibody fragment thereof isalso included in the antibody or antibody fragment of the presentinvention.

The number of amino acids which are deleted, substituted, insertedand/or added is one or more, and is not specifically limited, but it iswithin the range where deletion, substitution or addition is possible byknown methods such as the site-directed mutagenesis described inMolecular Cloning, Second Edition, Cold Spring Harbor Laboratory Press(1989); Current Protocols in Molecular Biology, John Wiley & Sons(1987-1997); Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad.Sci. USA, 79, 6409 (1982); Gene, 34, 315 (1985), Nucleic Acids Research,13, 4431 (1985); Proc. Natl. Acad. Sci. USA, 82, 488 (1985) or the like.For example, the number is preferably 1 to dozens, more preferably 1 to20, furthermore preferably 1 to 10, and most preferably 1 to 5.

The expression “one or more amino acid residue(s) is/are deleted,substituted, inserted and/or added” in the amino acid sequence of theabove antibody means the followings. That is, it means there isdeletion, substitution, insertion or addition of one or plural aminoacids at optional positions in the same sequence and in one or pluralamino acid sequences. Also, the deletion, substitution, insertion oraddition may occur at the same time and the amino acid which issubstituted, inserted or added may be either a natural type or anon-natural type.

The natural type amino acid includes L-alanine, L-asparagine, L-asparticacid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and thelike.

Preferable examples of mutually substitutable amino acids are shownbelow. The amino acids in the same group are mutually substitutable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine,t-butylalanine, cyclohexylalanine

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid, 2-aminosuberic acid

Group C: asparagine, glutamine

Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acid

Group E: proline, 3-hydroxyproline, 4-hydroxyproline

Group F: serine, threonine, homoserine

Group G: phenylalanine, tyrosine

The antibody fragment of the present invention includes Fab, F(ab′)₂,Fab′, a single chain antibody (scFv), a dimerized V region (diabody), adisulfide stabilized V region (dsFv), a peptide comprising CDR and thelike.

An Fab is an antibody fragment among fragments obtained by treating anIgG antibody molecule with a protease, papain (cleaved at an amino acidresidue at position 224 of the H chain), having a molecular weight ofabout 50,000 and having antigen binding activity, in which about a halfof the N-terminal side of H chain and the entire L chain are boundtogether through a disulfide bond.

The Fab of the present invention can be produced by treating themonoclonal antibody of the present invention which specificallyrecognizes human TIM-3 and binds to an amino acid sequence of theextracellular region or three-dimensional structure thereof with papain.Also, the Fab can be produced by inserting DNA encoding Fab of theantibody into an expression vector for prokaryote or an expressionvector for eukaryote, and introducing the vector into a prokaryote oreukaryote to express the Fab.

An F(ab′)₂ is an antibody fragment having a molecular weight of about100,000 and having antigen binding activity and comprising two Fabregions which are bound in the hinge position obtained by digesting thelower part of two disulfide bonds in the hinge region of IgG with anenzyme, pepsin.

The F(ab′)₂ of the present invention can be produced by treating themonoclonal antibody of the present invention which specificallyrecognizes human TIM-3 and binds to an amino acid sequence of theextracellular region or three-dimensional structure thereof with pepsin.Also, the F(ab′)₂ can be produced by binding Fab′ described below via athioether bond or a disulfide bond.

An Fab′ is an antibody fragment having a molecular weight of about50,000 and antigen binding activity, which is obtained by cleaving adisulfide bond at the hinge region of the above F(ab′)₂. The Fab′ of thepresent invention can be produced by treating the F(ab′)₂ of the presentinvention which specifically recognizes TIM-3 and binds to an amino acidsequence of the extracellular region or three-dimensional structurethereof with a reducing agent, such as dithiothreitol. Also, the Fab′can be produced by inserting DNA encoding Fab′ fragment of the antibodyinto an expression vector for prokaryote or an expression vector foreukaryote, and introducing the vector into a prokaryote or eukaryote toexpress the Fab′.

An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain VH andone chain VL are linked using an appropriate peptide linker (hereinafterreferred to as “P”) and is an antibody fragment having antigen bindingactivity.

The scFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of the monoclonal antibody of the present inventionwhich specifically recognizes human TIM-3 and binds to an amino acidsequence of the extracellular region or three-dimensional structurethereof, constructing DNA encoding scFv, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing the expression vector into a prokaryote oreukaryote to express the scFv.

A diabody is an antibody fragment wherein scFv is dimerized, is anantibody fragment having divalent antigen binding activity. In thedivalent antigen binding activity, two antigens may be the same ordifferent.

The diabody of the present invention can be produced by obtaining cDNAsencoding VH and VL of the monoclonal antibody of the present inventionwhich specifically recognizes human TIM-3 and binds to an amino acidsequence of the extracellular region or three-dimensional structurethereof, constructing DNA encoding scFv so that the length of the aminoacid sequence of the peptide linker is 8 or less residues, inserting theDNA into an expression vector for prokaryote or an expression vector foreukaryote, and then introducing the expression vector into a prokaryoteor eukaryote to express the diabody.

A dsFv is obtained by binding polypeptides in which one amino acidresidue of each of VH and VL is substituted with a cysteine residue viaa disulfide bond between the cysteine residues. The amino acid residueto be substituted with a cysteine residue can be selected based on athree-dimensional structure estimation of the antibody in accordancewith a known methods [Protein Engineering, 7, 697 (1994)].

The dsFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of the monoclonal antibody of the present inventionwhich specifically recognizes human TIM-3 and binds to an amino acidsequence of the extracellular region or three-dimensional structurethereof, constructing DNA encoding dsFv, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing the expression vector into a prokaryote oreukaryote to express the dsFv.

A peptide comprising CDR is constituted by including one or more regionsof CDRs of VH or VL. Peptide comprising plural CDRs can be bounddirectly or via an appropriate peptide linker.

The peptide comprising CDR of the present invention can be produced byconstructing DNA encoding CDRs of VH and VL of the monoclonal antibodyof the present invention which specifically recognizes human TIM-3 andbinds to an amino acid sequence of the extracellular region orthree-dimensional structure thereof, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing the expression vector into a prokaryote oreukaryote to express the peptide. The peptide comprising CDR can also beproduced by a chemical synthesis method such as Fmoc method or tBocmethod.

The monoclonal antibody of the present invention includes an antibodyconjugate in which the monoclonal antibody or the antibody fragment ofthe present invention which specifically recognizes human TIM-3 andbinds to an amino acid sequence of the extracellular region orthree-dimensional structure thereof is chemically or genetically boundto a radioisotope, an agent having a low molecular weight, an agenthaving a high molecular weight, a protein, a therapeutic antibody or thelike.

The antibody conjugate of the present invention can be produced bychemically conjugating a radioisotope, an agent having a low molecularweight, an agent having a high molecular weight, an adjuvant, a protein,a therapeutic antibody or the like to the N-terminal side or C-terminalside of an H chain or an L chain, adequate substituent or sidechain,sugarchain, and the like of the monoclonal antibody or the antibodyfragment of the present invention which specifically recognizes humanTIM-3 and binds to an amino acid sequence of the extracellular region orthree-dimensional structure thereof [Kotai Kogaku Nyumon, published byChijin Shokan (1994)].

Also, the antibody conjugate can be genetically produced by linking aDNA encoding the monoclonal antibody or the antibody fragment of thepresent invention which specifically recognizes human TIM-3 and binds toan amino acid sequence of the extracellular region or three-dimensionalstructure thereof to other DNA encoding a protein or a therapeuticantibody to be conjugated, inserting the DNA into a vector forexpression, and introducing the expression vector into an appropriatehost cell.

The radioisotope includes ¹³¹I, ¹²⁵I, ⁹⁰Y, ⁶⁴Cu, ¹⁹⁹Tc, ⁷⁷Lu, ²¹¹At andthe like. The radioisotope can directly be conjugated with the antibodyby Chloramine-T method or the like. Also, a substance chelating theradioisotope can be conjugated with the antibody. The chelating agentincludes 1-isothiocyanatobenzyl-3-methyldiethylene-triaminepentaaceticacid (MX-DTPA) and the like.

The agent having a low molecular weight includes an anti-tumor agentsuch as an alkylating agent, a nitrosourea agent, a metabolismantagonist, an antibiotic substance, an alkaloid derived from a plant, atopoisomerase inhibitor, an agent for hormonotherapy, a hormoneantagonist, an aromatase inhibitor, a P glycoprotein inhibitor, aplatinum complex derivative, an M-phase inhibitor and a kinase inhibitor[Rinsho Syuyo-gaku (Clinical Oncology), Gan to Kagakuryoho-Sha (1996)],a steroid agent such as hydrocortisone and prednisone, a nonsteroidalagent such as aspirin and indomethacin, immunomodulatory agent such asaurothiomalate, penicillamine, immuno-suppressing agent such ascyclophosphamide and azathioprine, anti-inflammatory agent such asanti-histamine agent, for example, chlorpheniramine maleate andclemastine [Ensho to Kouensho-Ryoho (Inflammation and Anti-inflammationTherapy), Ishiyaku Shuppann (1982)] and the like.

Examples of the antitumor agent include amifostine (Ethyol), cisplatin,dacarbazine (DTIC), dactinomycin, mecloretamin (nitrogen mustard),streptozocin, cyclophosphamide, iphosphamide, carmustine (BCNU),lomustine (CCNU), doxorubicin (adriamycin), epirubicin, gemcitabine(Gemsal), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide,methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine,bleomycin, daunomycin, peplomycin, estramustine, paclitaxel (Taxol),docetaxel (Taxotea), aldesleukin, asparaginase, busulfan, carboplatin,oxaliplatin, nedaplatin, cladribine, camptothecin,10-hydroxy-7-ethylcamptothecin (SN38), floxuridine, fludarabine,hydroxyurea, iphosphamide, idarubicin, mesna, irinotecan (CPT-11),nogitecan, mitoxantrone, topotecan, leuprolide, megestrol, melfalan,mercaptopurine, hydroxycarbamide, plicamycin, mitotane, pegasparagase,pentostatin, pipobroman, streptozocin, tamoxifen, goserelin,leuprorelin, flutamide, teniposide, testolactone, thioguanine, thiotepa,uracil mustard, vinorelbine, chlorambucil, hydrocortisone, prednisolone,methylprednisolone, vindesine, nimustine, semustine, capecitabine,Tomudex, azacytidine, UFT, oxaliplatin, gefitinib (Iressa), imatinib(STI 571), elrotinib, FMS-like tyrosine kinase 3 (Flt3) inhibitor,vascular endothelial growth facotr receptor (VEGFR) inhibitor,fibroblast growth factor receptor (FGFR) inhibitor, Epidermal GrowthFactor Receptor (EGFR) inhibitor such as Iressa and Tarceva, radicicol,17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine,all-trans-retinoic acid, thalidomide, anastrozole, fadrozole, letrozole,exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine,mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene(Targretin), tamoxifen, dexamethasone, progestin substances, estrogensubstances, anastrozole (Arimidex), Leuplin, aspirin, indomethacin,celecoxib, azathioprine, penicillamine, gold thiomalate,chlorpheniramine maleate, chlorpheniramine, clemastine, tretinoin,bexarotene, arsenic, voltezomib, allopurinol, calicheamicin, ibritumomabtiuxetan, Targretin, ozogamine, clarithromycin, leucovorin, ifosfamide,ketoconazole, aminoglutethimide, suramin, methotrexate, maytansinoid andderivatives thereof.

The method for conjugating the agent having low molecular weight withthe antibody includes a method in which the agent and an amino group ofthe antibody are conjugated through glutaraldehyde, a method in which anamino group of the agent and a carboxyl group of the antibody areconjugated through water-soluble carbodiimide, and the like.

The agent having a high molecular weight includes polyethylene glycol(hereinafter referred to as “PEG”), albumin, dextran, polyoxyethylene,styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer,hydroxypropylmethacrylamide, and the like. By binding these compoundshaving a high molecular weight to an antibody or antibody fragment, thefollowing effects are expected: (1) improvement of stability againstvarious chemical, physical or biological factors, (2) remarkableprolongation of half life in blood, (3) disappearance of immunogenicityor suppression of antibody production, and the like [Bioconjugate Drug,Hirokawa Shoten (1993)]. For example, the method for binding PEG to anantibody includes a method in which an antibody is allowed to react witha PEG-modifying reagent [Bioconjugate Drug, Hirokawa Shoten (1993)]. ThePEG-modifying reagent includes a modifying agent of ε-amino group oflysine (Japanese Published Unexamined Patent Application No. 178926/86),a modifying agent of a carboxyl group of aspartic acid and glutamic acid(Japanese Published Unexamined Patent Application No. 23587/81), amodifying agent of a guanidino group of arginine (Japanese PublishedUnexamined Patent Application No. 117920/90) and the like.

The immunostimulator may be any natural products known asimmunoadjuvants. Examples of an agent enhancing immunogen includeβ(1→3)glucan (lentinan, schizophyllan), α-galactosylceramide and thelike.

The protein includes a cytokine or a growth factor which activates aimmunocompetent cell, such as NK cell, macrophage or neutrophil, a toxicprotein, and the like.

Examples of the cytokine or the growth factor include interferon(hereinafter referred to as “IFN”)-α, IFN-β, IFN-γ, interleukin(hereinafter referred to as “IL”)-2, IL-12, IL-15, IL-18, IL-21, IL-23,granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), macrophage-colonystimulating factor (M-CSF) and the like.

The toxic protein includes ricin, diphtheria toxin, ONTAK and the like,and also includes a toxic protein wherein mutation is introduced into aprotein in order to control the toxicity.

The therapeutic antibody includes an antibody against an antigen inwhich apoptosis is induced by binding of the antibody, an antibodyagainst an antigen participating in formation of pathologic state oftumor, an antibody against an antigen regulating immunological functionand an antibody against an antigen relating to angiogenesis in thepathologic part.

The antigen in which apoptosis is induced by binding of the antibodyincludes cluster of differentiation (hereinafter “CD”) 19, CD20, CD21,CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77,CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86(B7.2), human leukocyte antigen (HLA)-Class II, epidermal growth factorreceptor (EGFR) and the like.

The antigen participating in formation of pathologic state of tumor orthe antigen regulating immunological function includes CD40, CD40ligand, B7 family molecule (CD80, CD86, CD274, B7-DC, B7-H2, B7-H3,B7-H4), ligand of B7 family molecule (CD28, CTLA-4, ICOS, PD-1, BTLA),OX-40, OX-40 ligand, CD137, tumor necrosis factor (TNF) receptor familymolecule (DR4, DR5, TNFR1, TNFR2), TNF-related apoptosis-inducing ligandreceptor (TRAIL) family molecule, receptor family of TRAIL familymolecule (TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4), receptor activator ofnuclear factor kappa B (RANK), RANK ligand, CD25, folic acid receptor 4,cytokine [IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforminggrowth factor (TGF) β, TNFα, etc.], receptors of these cytokines,chemokine (SLC, ELC, I-309, TARC, MDC, CTACK, etc.) and receptors ofthese chemokines.

The antigen for the antibody which inhibits angiogenesis in thepathologic part includes vascular endothelial growth factor (VEGF),angiopoietin, fibroblast growth factor (FGF), EGF, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF), erythropoietin(EPO), TGFβ, IL-8, Ephilin, SDF-1, receptors thereof and the like.

A fusion antibody with a protein or therapeutic antibody can be producedby linking a cDNA encoding a monoclonal antibody or antibody fragment toa cDNA encoding the protein, constructing a DNA encoding the fusionantibody, inserting the DNA into an expression vector for prokaryote oreukaryote, and then introducing the expression vector into a prokaryoteor eukaryote to express the fusion antibody.

In the case where the above antibody conjugate is used for the detectionmethod, method for quantitative determination, detection reagent,reagent for quantitative determination or diagnostic agent in thepresent invention, examples of the agent to which the monoclonalantibody or the antibody fragment of the present invention whichspecifically recognizes human TIM-3 and binds to an amino acid sequenceof the extracellular region or three-dimensional structure thereof isbound includes a label used in routine immunological detecting ormeasuring method.

The label includes enzymes such as alkaline phosphatase, peroxidase andluciferase, luminescent materials such as acridinium ester and lophine,fluorescent materials such as fluorescein isothiocyanate (FITC) andtetramethyl rhodamine isothiocyanate (RITC), and the like.

Furthermore, the present invention includes an agent for treating adisease relating to a TIM-3 positive cell, comprising the monoclonalantibody of the present invention or the antibody fragment thereof as anactive ingredient.

The disease relating to the TIM-3 positive cell is not limited, so longas it is a disease relating to a cell expressing TIM-3, such as cancer,autoimmune disease, and allergic disease.

The cancer includes blood cancer, breast cancer, uterine cancer,colorectal cancer, esophageal cancer, gastric cancer, ovarian cancer,lung cancer, renal cancer, rectal cancer, thyroid cancer, uterine cervixcancer, small intestinal cancer, prostate cancer and pancreatic cancer.Preferable examples of the cancer include blood cancer, esophagealcancer, gastric cancer, colorectal cancer, liver cancer and prostatecancer.

Examples of the blood cancer include acute myeloid leukemia (AML),chronic myeloid leukemia (CML), myelodysplasticsyndromes (MDS), multiplemyeloma, cutaneous T cell lymphoma (CTCL), peripheral T-cell lymphoma(PTCL), anaplastic large cell lymphoma (ALCL), acute lymphocyticleukemia (ALL), chronic lymphocytic leukemia (CLL), other lymphoidleukemia, NK cell lymphoma, Hodgkin lymphoma, non-Hodgkin's lymphomasuch as Burkitt's lymphoma, and the like.

Specific examples of the autoimmune diseases include rheumatoidarthritis, psoriasis, Crohn's disease, Ankylosing spondylitis, multiplesclerosis, type I diabetes, hepatitis, myocarditis, Sjogren's syndrome,autoimmune hemolytic anemia after transplant rejection, blisterspemphigoid, Graves disease, Hashimoto's thyroiditis, systemic lupuserythematosus (SLE), myasthenia gravis, pemphigus, pernicious anemia,and the like.

Examples of the allergic diseases include acute or chronic reactiveairway disease, bronchial asthma, atopic dermatitis, allergic rhinitis,urticaria, PIE syndrome, food allergies, hay fever, allergic nose,bronchial asthma, atopic dermatitis, anaphylactic shock and the like.

The therapeutic agent of the present invention comprises the abovemonoclonal antibody or the antibody fragment of the present invention asan active ingredient.

The therapeutic agent comprising the monoclonal antibody of the presentinvention or antibody fragment thereof, or conjugate thereof maycomprise only the antibody or antibody fragment thereof, or conjugatethereof as an active ingredient. It is generally preferred that thetherapeutic agent is prepared as a pharmaceutical preparation producedby an appropriate method well known in the technical field ofpharmaceutics, and by mixing it with one or more pharmaceuticallyacceptable carriers.

It is preferred to administer the therapeutic agent by the route that ismost effective for the treatment. Examples include oral administrationand parenteral administration, such as buccal, tracheal, rectal,subcutaneous, intramuscular or intravenous administration andintravenous administration is preferred.

The pharmaceutical preparation includes sprays, capsules, tablets,powders, granules, syrups, emulsions, suppositorys, injections,ointments, tapes and the like.

Although the dose or the frequency of administration varies depending onthe objective therapeutic effect, administration method, treatingperiod, age, body weight and the like, it is usually 10 μg/kg to 10mg/kg per day and per adult.

Further, the present invention relates to a method for immunologicallydetecting or measuring TIM-3, a reagent for immunologically detecting ormeasuring TIM-3, a method for immunologically detecting or measuring acell expressing TIM-3, and a diagnostic agent for diagnosing a diseaserelating to a TIM-3 positive cell, comprising the monoclonal antibody orthe antibody fragment of the present invention which specificallyrecognizes human TIM-3 and binds to an amino acid sequence of theextracellular region or three-dimensional structure thereof as an activeingredient.

In the present invention, the method for detecting or measuring theamount of TIM-3 may be any known method. For example, it includes animmunological detecting or measuring method.

The immunological detecting or measuring method is a method in which anantibody amount or an antigen amount is detected or determined using alabeled antigen or antibody. Examples of the immunological detecting ormeasuring method include radioactive substance-labeled immunoantibodymethod (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay(FIA), luminescent immunoassay, Western blotting method, physicochemicalmethod and the like.

The above disease relating to a TIM-3 positive cell can be diagnosed bydetecting or measuring a cell expressing TIM-3 by using the monoclonalantibody or antibody fragment of the present invention.

For the detection of the cell expressing the polypeptide, knownimmunological detection methods can be used, and an immunoprecipitationmethod, a fluorescent cell staining method, an immune tissue stainingmethod and the like are preferably used. Also, a fluorescent antibodystaining method using FMAT 8100 HTS system (Applied Biosystem) and thelike can be used.

In the present invention, the living body sample to be used fordetecting or measuring TIM-3 is not particularly limited, so long as ithas a possibility of containing a cell expressing TIM-3, such as tissuecells, blood, blood plasma, serum, pancreatic fluid, urine, fecalmatter, tissue fluid or culture fluid.

The diagnostic agent containing the monoclonal antibody of the presentinvention or antibody fragment thereof, or conjugate thereof may furthercontain a reagent for carrying out an antigen-antibody reaction or areagent for detection of the reaction depending on the desireddiagnostic method. The reagent for carrying out the antigen-antibodyreaction includes a buffer, a salt, and the like. The reagent fordetection includes a reagent generally used for the immunologicaldetecting or measuring method, such as labeled secondary antibody whichrecognizes the monoclonal antibody, antibody fragment thereof orconjugates thereof and substrate corresponding to the labeling and thelike.

A process for producing the antibody of the present invention, a methodfor treating the disease and a method for diagnosing the disease arespecifically described below.

1. Preparation Method of Monoclonal Antibody

(1) Preparation of Antigen

TIM-3 or a cell expressing TIM-3 as an antigen can be obtained byintroducing an expression vector comprising cDNA encoding a full lengthof TIM-3 or a partial length thereof is introduced into Escherichiacoli, yeast, an insect cell, an animal cell or the like. In addition,TIM-3 can be purified and obtained from various human tumor cell lines,human tissue and the like which express a large amount of TIM-3. Thetumor cell line and the tissue can be directly allowed to use asantigens. Furthermore, a synthetic peptide having a partial sequence ofthe TIM-3 can be prepared by a chemical synthesis method such as Fmocmethod or tBoc method and used as an antigen.

TIM-3 used in the present invention can be produced, for example, byexpressing a DNA encoding TIM-3 in a host cell using a method describedin Molecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989), Current Protocols in Molecular Biology,John Wiley & Sons (1987-1997) or the like according to the followingmethod.

Firstly, a recombinant vector is prepared by inserting a full lengthcDNA comprising the region encoding TIM-3 into downstream of a promoterof an appropriate expression vector. At this time, if necessary, a DNAfragment having an appropriate length containing a region encoding thepolypeptide prepared based on the full length cDNA may be used insteadof the above full length cDNA. Next, a transformant producingpolypeptide can be obtained by introducing the recombinant vector into ahost cell suitable for the expression vector.

The expression vector may be any one, so long as it can replicateautonomously in the host cell to be used or it can be integrated into achromosome comprising an appropriate promoter at such a position thatthe DNA encoding the polypeptide can be transcribed.

The host cell may be any one, so long as it can express the objectivegene. Examples include a microorganism which belongs to the generaEscherichia, such as Escherichia coli, yeast, an insect cell, an animalcell and the like.

When a prokaryote such as Escherichia coli is used as the host cell, itis preferred that the recombinant vector used in the present inventionis autonomously replicable in the prokaryote and comprising a promoter,a ribosome binding sequence, the DNA encoding TIM-3 and a transcriptiontermination sequence. The recombinant vector is not necessary to have atranscription termination sequence, but a transcription terminationsequence is preferably set just below the structural gene. Therecombinant vector may further comprise a gene regulating the promoter.

Also, the above recombinant vector is preferably a plasmid in which thespace between Shine-Dalgarno sequence, which is the ribosome bindingsequence, and the initiation codon is adjusted to an appropriatedistance (for example, 6 to 18 nucleotides).

Furthermore, the nucleotide sequence of the DNA encoding TIM-3 can besubstituted with another base so as to be a suitable codon forexpressing in a host cell, thereby improve the productivity of theobjective TIM-3.

Any expression vector can be used, so long as it can function in thehost cell to be used. Examples of the expression vector includes pBTrp2,pBTac1, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2(manufactured by Pharmacia), pSE280 (manufactured by Invitrogen),pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN),pKYP10 (Japanese Published Unexamined Patent Application No. 110600/83),pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1[Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA,82, 4306 (1985)], pBluescript II SK(−) (manufactured by Stratagene),pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)],pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)],pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), JapanesePublished Unexamined Patent Application No. 221091/85], pGKA2 [preparedfrom Escherichia coli IGKA2 (FERM BP-6798), Japanese PublishedUnexamined Patent Application No. 221091/85], pTerm2 (U.S. Pat. No.4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat. No. 5,160,735), pSupex,pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX(manufactured by Pharmacia), pET system (manufactured by Novagen),pME18SFL3 and the like.

Any promoter can be used, so long as it can function in the host cell tobe used. Examples include promoters derived from Escherichia coli, phageand the like, such as trp promoter (Ptrp), lac promoter, PL promoter, PRpromoter and T7 promoter and the like. Also, artificially designed andmodified promoters, such as a promoter in which two Ptrp are tandemlylinked, tac promoter, lacT7 promoter and letI promoter, can be used.

Examples of host cell include Escherichia coli XL1-Blue, Escherichiacoli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000,Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109,Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110,Escherichia coli NY49, Escherichia coli DH5α and the like.

Any introduction method of the recombinant vector can be used, so longas it is a method for introducing DNA into the host cell, and examplesinclude a method using a calcium ion described in Proc. Natl. Acad. Sci.USA, 69, 2110 (1972), Gene, 17, 107 (1982) and Molecular & GeneralGenetics, 168, 111 (1979) and the like.

When an animal cell is used as the host cell, any expression vector canbe used, so long as it can function in the animal cell. Examples includepcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese PublishedUnexamined Patent Application No. 22979/91; Cytotechnology, 3, 133(1990)], pAS3-3 (Japanese Published Unexamined Patent Application No.227075/90), pCDM8 [Nature, 329, 840, (1987)], pcDNAI/Amp (manufacturedby Invitrogen), pcDNA3.1 (manufactured by Invitrogen), pREP4(manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307(1987)], pAGE210, pME18SFL3, pKANTEX93 (WO 97/10354) and the like.

Any promoter can be used, so long as it can function in an animal cell.Examples include a promoter of immediate early (1E) gene ofcytomegalovirus (CMV), SV40 early promoter, a promoter of retrovirus, ametallothionein promoter, a heat shock promoter, SRα promoter, Molonymurine leukemia virus promoter or enhancer, and the like. Also, theenhancer of the IE gene of human CMV can be used together with thepromoter.

The host cell includes human Namalwa leukemia cell, monkey COS cell,Chinese hamster ovary (CHO) cell [Journal of Experimental Medicine, 108,945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55,513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115(1996); Radiation Research, 148, 260 (1997); Proc. Natl. Acad. Sci. USA,77, 4216 (1980); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Cell, 6,121 (1975); Molecular Cell genetics, Appendix I, II (pp. 883-900)],CHO/DG44, CHO-K1 (ATCC accession NO: CCL-61), DUkXB11 (ATCC accessionNO:CCL-9096), Pro-5 (ATCC accession NO: CCL-1781), CHO-S (LifeTechnologies, Cat#11619), Pro-3, rat myeloma cell YB2/3HL.P2.G11.16Ag.20(also called as YB2/0), mouse myeloma cell NSO, mouse myeloma cellSP2/0-Ag14, Syrian hamster cell BHK or HBT5637 (Japanese PublishedUnexamined Patent Application No. 299/88) and the like.

Any introduction method of the recombinant vector can be used, so longas it is a method for introducing DNA into an animal cell, and examplesinclude electroporation [Cytotechnology, 3, 133 (1990)], the calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)], and the like.

TIM-3 can be produced by culturing the transformant derived from amicroorganism, an animal cell or the like having a recombinant vectorcomprising the DNA encoding TIM-3 in a medium to form and accumulateTIM-3 in the culture, and recovering it from the culture. The method forculturing the transformant in the medium is carried out according to theusual method used in culturing of hosts.

When TIM-3 is expressed in a cell derived from eukaryote, TIM-3 to whichsugars or sugar chains bind can be obtained.

When a microorganism transformed with a recombinant vector containing aninducible promoter is cultured, an inducer can be added to the medium,if necessary.

For example, isopropyl-β-D-thiogalactopyranoside or the like can beadded to the medium when a microorganism transformed with a recombinantvector using lac promoter is cultured; or indoleacrylic acid or the likecan be added thereto when a microorganism transformed with a recombinantvector using trp promoter is cultured.

When a transformant obtained using an animal cell as the host cell iscultured, the medium includes generally used RPMI 1640 medium [TheJournal of the American Medical Association, 199, 519 (1967)], Eagle'sMEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium[Virology, 8, 396 (1959)] and 199 medium [Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)], Iscove's modified Dulbecco'smedium (IMDM), the media to which fetal calf serum, etc. is added, andthe like. Culture is carried out generally at a pH of 6 to 8 and 30 to40° C. for 1 to 7 days in the presence of 5% CO₂. If necessary, anantibiotic such as kanamycin or penicillin can be added to the mediumduring the culturing.

Regarding the expression method of the gene encoding TIM-3, in additionto direct expression, secretory production, fusion protein expressionand the like can be carried out according to the method described inMolecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989).

The process for producing TIM-3 includes a method of intracellularexpression in a host cell, a method of extracellular secretion from ahost cell, a method of producing on a host cell outer membrane, and thelike. The appropriate method can be selected by changing the host cellused and the structure of the TIM-3 produced.

When the TIM-3 is produced in a host cell or on a host cell membraneouter envelope, TIM-3 can be positively secreted extracellularly inaccordance with the method of Paulson et al. [J. Biol. Chem., 264, 17619(1989)], the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227(1989), Genes Develop., 4, 1288 (1990)], the methods described inJapanese Published Unexamined Patent Application No. 336963/93 and WO94/23021, and the like.

Also, the production amount of TIM-3 can be increased in accordance withthe method described in Japanese Published Unexamined Patent ApplicationNo. 227075/90 utilizing a gene amplification system using adihydrofolate reductase gene.

The resulting TIM-3 can be isolated and purified, for example, asfollows.

When TIM-3 is intracellularly expressed in a dissolved state, the cellsafter culturing are recovered by centrifugation, suspended in an aqueousbuffer and then disrupted using ultrasonicator, French press, MantonGaulin homogenizer, dynomill or the like to obtain a cell-free extract.

The cell-free extract is centrifuged to obtain a supernatant, and apurified preparation can be obtained by subjecting the supernatant to ageneral protein isolation and purification techniques such as solventextraction; salting out with ammonium sulfate etc.; desalting;precipitation with an organic solvent; anion exchange chromatographyusing a resin such as diethylaminoethyl (DEAE)-sepharose, DIAION HPA-75(manufactured by Mitsubishi Chemical); cation exchange chromatographyusing a resin such as S-Sepharose FF (manufactured by Pharmacia);hydrophobic chromatography using a resin such as butyl-Sepharose orphenyl-Sepharose; gel filtration using a molecular sieve; affinitychromatography; chromatofocusing; electrophoresis such as isoelectricfocusing; and the like which may be used alone or in combination.

When TIM-3 is expressed intracellularly by forming an inclusion body,the cells are recovered, disrupted and centrifuged in the same manner,and the inclusion body of TIM-3 are recovered as a precipitationfraction. The recovered inclusion body of the TIM-3 protein issolubilized with a protein denaturing agent. The protein is made into anormal three-dimensional structure by diluting or dialyzing thesolubilized solution, and then a purified preparation of polypeptide isobtained by the same isolation purification method as above.

When TIM-3 or the derivative such as a glycosylated product is secretedextracellularly, TIM-3 or the derivative such as a glycosylated productcan be recovered from the culture supernatant. That is, the culture istreated by a method such as centrifugation in the same manner as aboveto obtain a soluble fraction, a purified preparation of TIM-3 can beobtained from the soluble fraction by the same isolation purificationmethod as above.

Also, TIM-3 used in the present invention can be produced by a chemicalsynthesis method, such as Fmoc method or tBoc method. Also, it can bechemically synthesized using a peptide synthesizer manufactured byAdvanced ChemTech, Perkin-Elmer, Pharmacia, Protein TechnologyInstrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, or thelike.

(2) Immunization of Animal and Preparation of Antibody-Producing Cellfor Fusion

A mouse, rat or hamster and the like which is 3 to 20-weeks-old isimmunized with the antigen prepared in the above (1), andantibody-producing cells within the spleen, lymph node or peripheralblood of the animal are collected. Also, when the increase of asufficient titer in the above animal is not recognized due to lowimmunogenicity, a TIM-3 knockout mouse may by used as an animal to beimmunized.

The immunization is carried out by administering the antigen to theanimal through subcutaneous, intravenous or intraperitoneal injectiontogether with an appropriate adjuvant (for example, complete Freund'sadjuvant, combination of aluminum hydroxide gel with pertussis vaccine,or the like). When the antigen is a partial peptide, a conjugate isproduced with a carrier protein such as BSA (bovine serum albumin), KLH(keyhole limpet hemocyanin) or the like, which is used as the antigen.

The administration of the antigen is carried out 5 to 10 times every oneweek or every two weeks after the first administration. On the 3rd to7th day after each administration, a blood sample is collected from thefundus of the eye, the reactivity of the serum with the antigen istested, for example, by enzyme immunoassay [Antibodies—A LaboratoryManual, Cold Spring Harbor Laboratory (1988)] or the like. An animalshowing a sufficient antibody titer in their sera against the antigenused for the immunization is used as the source of antibody-producingcells for fusion.

Three to seven days after final administration of the antigen, tissuecontaining the antibody-producing cells such as the spleen is excisedfrom the immunized animal to collect the antibody-producing cells. Whenthe spleen cells are used, the spleen is cut out and loosened, followedby centrifugation. Then, antibody-producing cells for fusion areobtained by removing erythrocytes.

(3) Preparation of Myeloma Cell

An established cell line obtained from mouse is used as myeloma cells.Examples include 8-azaguanine-resistant mouse (derived from BALB/c)myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiologyand Immunology, 18, 1 (1978)], P3-NS1/1-Ag41 (NS-1) [European J.Immunology, 6, 511 (1976)], SP2/0-Ag14 (SP-2) [Nature, 276, 269 (1978)],P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)], P3-X63-Ag8 (X63)[Nature, 256, 495 (1975)] and the like.

The myeloma cells are subcultured in a normal medium [a medium in whichglutamine, 2-mercaptoethanol, gentamicin, FBS and 8-azaguanine are addedto RPMI-1640 medium] and they are subcultured in the normal medium 3 or4 days before cell fusion to ensure the cell number of 2×10⁷ or more onthe day for fusion.

(4) Cell Fusion and Preparation of Hybridoma for Producing MonoclonalAntibody

The antibody-producing cells for fusion obtained by the above (2) andmyeloma cells obtained by the above (3) were sufficiently washed with aminimum essential medium (MEM) or PBS (1.83 g of disodium hydrogenphosphate, 0.21 g of potassium dihydrogen phosphate, 7.65 g of sodiumchloride, 1 liter of distilled water, pH 7.2) and mixed to give a ratioof the antibody-producing cells: the myeloma cells=5 to 10:1, followedby centrifugation. Then, the supernatant is discarded.

The precipitated cell group is sufficiently loosened. After looseningthe precipitated cell, the mixture of polyethylene glycol-1000(PEG-1000), MEM and dimethylsulfoxide is added to the cell understirring at 37° C. In addition, 1 to 2 mL of MEM medium is added severaltimes every one or two minutes, and MEM is added to give a total amountof 50 mL. After centrifugation, the supernatant is discarded. After thecells are gently loosened, the cells are gently suspended in HAT medium[a normal medium containing hypoxanthine, thymidine and aminopterin].The suspension is cultured in a 5% CO₂ incubator for 7 to 14 days at 37°C.

After the culturing, a portion of the culture supernatant is sampled anda hybridoma which is reactive to an antigen containing TIM-3 and is notreactive to an antigen not containing TIM-3 is selected by binding assayas described below. Then, cloning is carried out twice by a limitingdilution method [Firstly, HT medium (HAT medium from which aminopterinis removed) is used, and secondly, the normal medium is used], and ahybridoma which stably shows a high antibody titer is selected as themonoclonal antibody-producing hybridoma.

(5) Preparation of Purified Monoclonal Antibody

The hybridoma cells producing a monoclonal antibody obtained by theabove (4) are administered by intraperitoneal injection into 8- to10-week-old mice or nude mice treated with 0.5 mL of pristane(2,6,10,14-tetramethylpentadecane (pristane) is intraperitoneallyadministered, followed by feeding for 2 weeks). The hybridoma developsascites tumor in 10 to 21 days. The ascitic fluid is collected from themice, centrifuged to remove solids, subjected to salting out with 40 to50% ammonium sulfate and then precipitated by caprylic acid, passedthrough a DEAE-Sepharose column, a protein A column or a gel filtrationcolumn to collect an IgG or IgM fraction as a purified monoclonalantibody.

Furthermore, a monoclonal antibody-producing hybridoma obtained by theabove (4) is cultured in RPMI1640 medium containing 10% FBS or the likeand the supernatant is removed by centrifugation. The precipitated cellsare suspended in Hybridoma-SFM medium and cultured for 3 to 7 days. Thepurified monoclonal antibody can be obtained by centrifusing theobtained cell suspension, followed by purification from the resultingsupernatant with Protein A column or Protein G column to collect the IgGfractions. Additionally, Hybridoma-SFM medium can contain 5% DIGO GF21.

The subclass of the antibody can be determined using a subclass typingkit by enzyme immunoassay. The amount of the protein can be determinedby the Lowry method or from the absorbance at 280 nm.

(6) Selection of Monoclonal Antibody

Selection of monoclonal antibody is carried out by the following bindingassay using an enzyme immunoassay method and inhibition assay ofintracellular uptake of amino acids.

(6-a) Binding Assay

As the antigen, a gene-introduced cell obtained by introducing anexpression vector containing a cDNA encoding TIM-3 obtained in (1) intoEscherichia coli, yeast, an insect cell, an animal cell or the like, arecombinant protein, or a purified polypeptide or partial peptideobtained from a human tissue is used. When the antigen is a partialpeptide, a conjugate is prepared with a carrier protein such as BSA orKLH is prepared and is used.

After making these antigens into a solid layer by dispensing in a96-well plate, a substance to be tested such as serum, a culturesupernatant of a hybridoma or a purified monoclonal antibody isdispensed therein as the primary antibody and allowed to react. Afterthoroughly washing with PBS, PBS-Tween and the like, ananti-immunoglobulin antibody labeled with biotin, an enzyme, achemiluminescent material, a radiation compound or the like is dispensedtherein as the secondary antibody and allowed to react. After thoroughlywashing with PBS-Tween, the reaction appropriate to the label of thesecondary antibody is carried out to select a monoclonal antibody whichspecifically reacts with the antigen.

In addition, a monoclonal antibody which competes with the anti-TIM-3monoclonal antibody of the present invention in the binding of anextracellular region of human TIM-3 can be obtained by adding a subjectantibody to the above binding assay system to allow to react. Namely, amonoclonal antibody which competes with the obtained monoclonal antibodyin the binding of an amino acid sequence of the extracellular region ofhuman TIM-3 or a three-dimensional structure thereof can be obtained byscreening a subject antibody which inhibits the binding of themonoclonal antibody upon adding to the antibody.

Furthermore, an antibody which binds to the same epitope to which themonoclonal antibody of the present invention which binds to an aminoacid sequence of the extracellular region of TIM-3 or three-dimensionalstructure thereof can be obtained by identifying an epitope of theantibody obtained by the above binding assay system, preparing a partialsynthetic peptide of the epitope or a synthetic peptide which mimics thethree-dimensional structure of the epitope and immunizing with thepeptide.

(6-b) Kinetic Analysis with Biacore

The kinetics between an antigen and a test substance is measured usingBiacore T100 and then the obtained results are analyzed using analysissoftware accompanied with the apparatus. After anti-mouse IgG antibodyis immobilized onto to a CM 5 sensor chip by an amine coupling method, atest substance such as culture supernatant of a hybridoma, a purifiedmonoclonal antibody is allowed to flow and bind at an appropriateamount, and an antigen at plural known concentrations is further allowedto flow. And then, the binding and dissociation are measured.

Using the obtained data and the software accompanied with the apparatus,the kinetics analysis is carried out using the 1:1 binding model toobtain necessary parameters. Otherwise, after human TIM-3 is immobilizedonto the sensor chip by an amino coupling method, a purified monoclonalantibody is allowed to flow at plural known concentrations followed bymeasuring the binding and dissociation. Using the obtained data and thesoftware accompanied with the apparatus, the kinetics analysis iscarried out using bivalent analyte model to obtain necessary parameters.

2. Preparation of Recombinant Antibody

As production examples of recombinant antibodies, processes forproducing a human chimeric antibody and a human CDR grafted antibody areshown below.

(1) Construction of Vector for Expression of Recombinant Antibody

A vector for expression of recombinant antibody is an expression vectorfor animal cell into which DNAs encoding CH and CL of a human antibodyhave been inserted, and is constructed by cloning each of DNAs encodingCH and CL of a human antibody into an expression vector for animal cell.

The C region of a human antibody may be used CH and CL of any humanantibody. Examples include CH belonging to γ1 subclass, CL belonging toκ class, and the like. As the DNAs encoding CH and CL of a humanantibody, the cDNA may be generally used and a chromosomal DNAcomprising an exon and an intron can be also used.

As the expression vector for animal cell, any expression vector can beused, so long as a gene encoding the C region of a human antibody can beinserted thereinto and expressed therein. Examples include pAGE107[Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)],pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78,1527 (1980], pSG1bd2-4 [Cytotechnol., 4, 173 (1990)], pSE1UK1Sed1-3[Cytotechnol., 13, 79 (1993)] and the like.

Examples of a promoter and enhancer used for an expression vector foranimal cell include an SV40 early promoter [J. Biochem., 101, 1307(1987)], a Moloney mouse leukemia virus LTR [Biochem. Biophys. Res.Commun., 149, 960 (1987)], an immunoglobulin H chain promoter [Cell, 41,479 (1985)] and enhancer [Cell, 33, 717 (1983)] and the like.

The vector for expression of recombinant antibody may be either of atype in which a gene encoding an antibody H chain and a gene encoding anantibody L chain exist on separate vectors or of a type in which bothgenes exist on the same vector (tandem type). In respect of easiness ofconstruction of a vector for expression of recombinant antibody,easiness of introduction into animal cells, and balance between theexpression amounts of antibody H and L chains in animal cells, a tandemtype of the vector for expression of recombinant antibody is morepreferred [J. Immunol. Methods, 167, 271 (1994)]. Examples of the tandemtype of the vector for expression of recombinant antibody includepKANTEX93 (WO 97/10354), pEE18 [Hybridoma, 17, 559 (1998)], and thelike.

(2) Obtaining of cDNA Encoding V Region of Antibody Derived fromNon-Human Animal And Analysis of Amino Acid Sequence

Obtaining of cDNAs encoding VH and VL of a non-human antibody andanalysis of amino acid sequence are carried out as follows.

mRNA is extracted from hybridoma cells producing a non-human antibody tosynthesize cDNA. The synthesized cDNA is cloned into a vector such as aphage or a plasmid, to prepare a cDNA library.

Each of a recombinant phage or recombinant plasmid containing cDNAencoding VH or VL is isolated from the library using DNA encoding a partof the C region or V region of a mouse antibody as the probe. The fulllength of the nucleotide sequences of VH and VL of a mouse antibody ofinterest on the recombinant phage or recombinant plasmid are determined,and the full length of the amino acid sequences of VH and VL are deducedfrom the nucleotide sequences, respectively.

Examples of the non-human animal for preparing a hybridoma cell whichproduces a non-human antibody include mouse, rat, hamster, rabbit or thelike. Any animals can be used so long as a hybridoma cell can beproduced therefrom.

Examples of the method for preparing total RNA from a hybridoma cellinclude a guanidine thiocyanate-cesium trifluoroacetate method [Methodsin Enzymol., 154, 3 (1987)], the use of a kit such as RNA easy kit(manufactured by Qiagen) and the like.

Examples of the method for preparing mRNA from total RNA include anoligo (dT) immobilized cellulose column method [Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989)], a method using a kit such as Oligo-dT30<Super>mRNA PurificationKit (manufactured by Takara Bio) and the like. Additionary, examples ofthe method for preparing mRNA include method using a kit Fast Track mRNAIsolation kit (manufactured by Invitrogen) or QuickPrep mRNAPurification Kit (manufactured by Pharmacia) and the like.

Examples of the method for synthesizing cDNA and preparing a cDNAlibrary include known methods [Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press (1989); CurrentProtocols in Molecular Biology, Supplement 1, John Wiley & Sons(1987-1997)]; a method using a kit such as Super Script Plasmid Systemfor cDNA Synthesis and Plasmid Cloning (manufactured by Invitrogen),ZAP-cDNA Synthesis Kit (manufactured by Stratagene), etc.; and the like.

The vector into which the synthesized cDNA using mRNA extracted from ahybridoma cell as the template is inserted for preparing a cDNA librarymay be any vector, so long as the cDNA can be inserted. Examples includeZAP ExPress [Strategies, 5, 58 (1992)], pBluescript II SK(+) [NucleicAcids Research, 17, 9494 (1989)], λZAPII (manufactured by Stratagene),λgt10 and λgt11 [DNA Cloning: A Practical Approach, I, 49 (1985)],Lambda BlueMid (manufactured by Clontech), λExCell and pT7T3-18U(manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)],pUC18 [Gene, 33, 103 (1985)], and the like.

Any Escherichia coli for introducing the cDNA library constructed by aphage or plasmid vector may be used, so long as the cDNA library can beintroduced, expressed and maintained. Examples include XL1-Blue MRF′[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088 andY1090 [Science, 222: 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)],K802 [J. Mol. Biol., 16, 118 (1966)], JM105 [Gene, 38, 275 (1985)], andthe like.

A colony hybridization or plaque hybridization method using an isotope-or fluorescence-labeled probe may be used for selecting cDNA clonesencoding VH or VL of a non-human antibody or the like from the cDNAlibrary [Molecular Cloning, A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1989)].

Also, the cDNAs encoding VH and VL can be prepared through polymerasechain reaction (hereinafter referred to as “PCR”; Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989); Current Protocols in Molecular Biology, Supplement 1, John Wiley& Sons (1987-1997)) by preparing primers and using cDNA prepared frommRNA or a cDNA library as the template.

The nucleotide sequence of the cDNA can be determined by digesting thecDNA selected with appropriate restriction enzymes and the like, cloningthe fragments into a plasmid such as pBluescript SK(−) (manufactured byStratagene), carrying out the reaction by a usually used nucleotidesequence analyzing method. For example, a nucleotide sequence analyzingmethod is carried out by using an automatic nucleotide sequence analyzersuch as ABI PRISM3700 (manufactured by PE Biosystems) and A.L.F. DNAsequencer (manufactured by Pharmacia) after a reaction such as thedideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)].

Whether the obtained cDNAs encode the full amino acid sequences of VHand VL of the antibody containing a secretory signal sequence can beconfirmed by estimating the full length of the amino acid sequences ofVH and VL from the determined nucleotide sequence and comparing themwith the full length of the amino acid sequences of VH and VL of knownantibodies [A.L.F. DNA sequencer, US Dept. Health and Human Services(1991)].

With regard to the full amino acid sequences of VH and VL of theantibody containing a secretory signal sequence, the length of thesecretory signal sequence and N-terminal amino acid sequence can beestimated by comparing them with the full length of the amino acidsequences of VH and VL of known antibodies [A.L.F. DNA sequencer, USDept. Health and Human Services (1991)], and furthermore the subgroup towhich they belong can be determined. In addition, the amino acidsequence of each CDR of VH and VL can be determined by comparing themwith the full length of the amino acid sequences of VH and VL of knownantibodies [A.L.F. DNA sequencer, US Dept. Health and Human Services(1991)].

Moreover, the novelty of the full length of the amino acid sequence ofVH and VL can be examined by carrying out a homology search withsequences in any database, for example, SWISS-PROT, PIR-Protein or thelike using the obtained full length of the amino acid sequences of VHand VL, for example, according to the BLAST method [J. Mol. Biol., 215,403 (1990)] or the like.

(3) Construction of Vector for Expression of Human Chimeric Antibody

cDNA encoding each of VH and VL of antibody of non-human animal iscloned in the upstream of genes encoding CH or CL of human antibody ofvector for expression of recombinant antibody mentioned in the above (1)to thereby construct a vector for expression of human chimeric antibody.

In order to ligate cDNA comprising a nucleotide sequence of 3′-terminalof VH or VL of antibody of non-human animal and a nucleotide sequence of5′-terminal of CH or CL of human antibody, each cDNA encoding VH or VLof a non-human animal antibody is prepared so as to encodes appropriateamino acids and have an appropriate recognition sequence of arestriction enzyme at a linkage portion.

The prepared cDNAs encoding VH and VL of antibody are respectivelycloned so that each of them is expressed in an appropriate form in theupstream of gene encoding CH or CL of the human antibody of the vectorfor expression of the human CDR-grafted antibody mentioned in the above(1) to construct a vector for expression of human chimeric antibody.

In addition, cDNA encoding VH or VL of a non-human animal antibody isamplified by PCR using a synthetic DNA having a recognition sequence ofan appropriate restriction enzyme at both ends and each of them iscloned to the vector for expression of recombinant antibody obtained inthe above (1).

(4) Construction of cDNA Encoding V Region of Human CDR-Grafted Antibody

cDNAs encoding VH or VL of a human CDR-grafted antibody can be obtainedas follows.

Amino acid sequences of framework region (hereinafter referred to as“FR”) in VH or VL of a human antibody to which amino acid sequences ofCDRs in VH or VL of an antibody derived from a non-human animal antibodyare transplanted are respectively selected. Any amino acid sequences ofFR of a human antibody can be used, so long as they are derived fromhuman.

Examples include amino acid sequences of FRs of human antibodiesregistered in database such as Protein Data Bank or the like, and aminoacid sequences common to subgroups of FRs of human antibodies [Sequencesof Proteins of Immunological Interest, US Dept. Health and HumanServices (1991)], and the like. In order to inhibit the decrease in thebinding activity of the antibody, amino acid sequences having highhomology (at least 60% or more) with the amino acid sequence of FR in VHor VL of the original antibody is selected.

Then, amino acid sequences of CDRs of the original antibody are graftedto the selected amino acid sequence of FR in VH or VL of the humanantibody, respectively, to design each amino acid sequence of VH or VLof a humanized antibody. The designed amino acid sequences are convertedto DNA sequences by considering the frequency of codon usage found innucleotide sequences of genes of antibodies [Sequence of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991)], andthe DNA sequence encoding the amino acid sequence of VH or VL of ahumanized antibody is designed.

Based on the designed nucleotide sequences, several synthetic DNAshaving a length of about 100 nucleotides are synthesized, and PCR iscarried out using them. In this case, it is preferred that 6 syntheticDNAs per each of the H chain and the L chain are designed in view of thereaction efficiency of PCR and the lengths of DNAs which can besynthesized.

Furthermore, the cDNA encoding VH or VL of a human CDR-grafted antibodycan be easily cloned into the vector for expression of human CDR-graftedantibody constructed in (1) by introducing the recognition sequence ofan appropriate restriction enzyme to the 5′ terminal of the syntheticDNAs existing on the both ends.

Otherwise, it can be carried out using a synthetic DNA as one DNAencoding each of the full-length H chain and the full-length L chainbased on the designed DNA sequence.

After the PCR, an amplified product is cloned into a plasmid such aspBluescript SK (−) (manufactured by Stratagene) or the like, and thenucleotide sequence is determined according to a method similar to themethod described in (2) to obtain a plasmid having a DNA sequenceencoding the amino acid sequence of VH or VL of a desired humanizedantibody.

(5) Modification of Amino Acid Sequence of V Region of Human CDR-GraftedAntibody

It is known that when a human CDR-grafted antibody is produced by simplygrafting only CDRs in VH and VL of an antibody derived from a non-humananimal into FRs of VH and VL of a human antibody, its antigen bindingactivity is lower than that of the original antibody derived from anon-human animal [BIO/TECHNOLOGY, 9, 266 (1991)].

In human CDR-grafted antibodies, among the amino acid sequences of FRsin VH and VL of a human antibody, an amino acid residue which directlyrelates to binding to an antigen, an amino acid residue which interactswith an amino acid residue in CDR, and an amino acid residue whichmaintains the three-dimensional structure of an antibody and indirectlyrelates to binding to an antigen are identified and modified to an aminoacid residue which is found in the original non-humanized antibody tothereby increase the antigen binding activity which has been decreased.

In order to identify the amino acid residues relating to the antigenbinding activity in FR, constructing the three-dimensional structure ofan antibody and analyzing can be done by X-ray crystallography [J. Mol.Biol., 112, 535 (1977)], computer-modeling [Protein Engineering, 7, 1501(1994)] or the like. In addition, modified human CDR-grafted antibodyhaving sufficient binding activity against antigen can be obtained byvarious attempts, such as producing several modified antibodies of eachantibody and examining their binding activities.

The modification of the amino acid sequence of FR in VH and VL of ahuman antibody can be accomplished using various synthetic DNA formodification according to PCR as described in (4). With regard to theamplified product obtained by the PCR, the nucleotide sequence isdetermined according to the method as described in (2) so that whetherthe objective modification has been carried out is confirmed.

(6) Construction of Vector for Expression of Human CDR-Grafted Antibody

A vector for expression of human CDR-grafted antibody can be constructedby cloning each cDNA encoding VH or VL of a constructed recombinantantibody into upstream of each gene encoding CH or CL of the humanantibody in the vector for expression of recombinant antibody asdescribed in (1).

For example, when recognizing sequences of an appropriate restrictionenzymes are introduced to the 5′-terminal of synthetic DNAs positionedat both ends among synthetic DNAs used in the construction of VH or VLof the human CDR-grafted antibody in (4) and (5), cloning can be carriedout so that they are expressed in an appropriate form in the upstream ofeach gene encoding CH or CL of the human antibody in the vector forexpression of a human CDR-grafted antibody as described in (1).

(7) Transient Expression of Recombinant Antibody

In order to efficiently evaluate the antigen binding activity of varioushuman CDR-grafted antibodies produced, the recombinant antibodies can beexpressed transiently using the vector for expression of antibody asdescribed in (3) and (6) or the modified expression vector thereof.

Any cell can be used as a host cell, so long as the host cell canexpress a recombinant antibody. For example, COS-7 cell (ATCC CRL1651)is used in view of its high expression amount [Methods in Nucleic AcidsRes., CRC Press, 283 (1991)].

Examples of the method for introducing the expression vector into COS-7cell include a DEAE-dextran method [Methods in Nucleic Acids Res., CRCPress, 283 (1991)], a lipofection method [Proc. Natl. Acad. Sci. USA,84, 7413 (1987)], and the like.

After introduction of the expression vector, the expression amount andantigen binding activity of the recombinant antibody in the culturesupernatant can be determined by the enzyme immunoassay [MonoclonalAntibodies—Principles and practice, Third edition, Academic Press(1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory(1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific(1987)] and the like.

(8) Obtaining Transformant which Stably Expresses Recombinant Antibodyand Preparation of Recombinant Antibody

A transformant which stably expresses a recombinant antibody can beobtained by introducing the vector for expression of recombinantantibody described in (3) and (6) into an appropriate host cell.

Examples of the method for introducing the expression vector into a hostcell include electroporation [Japanese Published Unexamined PatentApplication No. 257891/90, Cytotechnology, 3, 133 (1990)] and the like.

As the host cell into which a vector for expression of a recombinantantibody is introduced, any cell can be used, so long as it is a hostcell which can produce the recombinant antibody. Examples include CHO-K1(ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S(Life Technologies, Cat #11619), rat myeloma cell YB2/3HL.P2.G11.16Ag.20(also referred to as YB2/0), mouse myeloma cell NSO, mouse myeloma cellSP2/0-Ag14 (ATCC No. CRL1581), mouse P3×63-Ag8653 cell (ATCC No.CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafterreferred to as “dhfr”) is defective [Proc. Natl. Acad. Sci. U.S.A., 77,4216 (1980)], lection resistance-acquired Lec13 [Somatic Cell andMolecular genetics, 12, 55 (1986)], CHO cell in whichα-1,6-fucosyltransaferse gene is defected (WO 2005/35586, WO 02/31140),rat YB2/3HL.P2.G11.16Ag.20 cell (ATCC No. CRL1662), and the like.

In addition, host cells in which activity of a protein such as an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,a protein such as an enzyme relating to the modification of a sugarchain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in a complex typeN-glycoside-linked sugar chain, or a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body isdecreased or deleted, preferably CHO cell in whichα1,6-fucosyltransferase gene is defected as described in WO2005/35586,WO02/31140 or the like, can also be used.

After introduction of the expression vector, transformants which expressa recombinant antibody stably are selected by culturing in a medium foranimal cell culture containing an agent such as G418 sulfate(hereinafter referred to as “G418”) or the like (Japanese PublishedUnexamined Patent Application No. 257891/90).

Examples of the medium for animal cell culture include RPMI1640 medium(manufactured by Invitrogen), GIT medium (manufactured by NihonPharmaceutical), EX-CELL301 medium (manufactured by JRH), IMDM medium(manufactured by Invitrogen), Hybridoma-SFM medium (manufactured byInvitrogen), media obtained by adding various additives such as FBS tothese media, and the like.

The recombinant antibody can be produced and accumulated in a culturesupernatant by culturing the obtained transformants in a medium. Theexpression amount and antigen binding activity of the recombinantantibody in the culture supernatant can be measured by ELISA or thelike. Also, in the transformant, the expression amount of therecombinant antibody can be increased by using DHFR amplification systemor the like according to the method disclosed in Japanese PublishedUnexamined Patent Application No. 257891/90.

The recombinant antibody can be purified from the culture supernatant ofthe transformant by using a protein A column [MonoclonalAntibodies—Principles and practice, Third edition, Academic Press(1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory(1988)]. In addition, the recombinant antibody can be purified by acombination of the methods for purification such as gel filtration,ion-exchange chromatography, ultrafiltration and the like.

The molecular weight of the H chain or the L chain of the purifiedrecombinant antibody or the antibody molecule as a whole is determinedby polyacrylamide gel electrophoresis (hereinafter referred to as“SDS-PAGE”) [Nature, 227, 680 (1970)], Western blotting [MonoclonalAntibodies—Principles and practice, Third edition, Academic Press(1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory(1988)], and the like.

3. Activity Evaluation of the Monoclonal Antibody or Antibody Fragment

The activity of the purified monoclonal antibody or antibody fragment ofthe present invention can be evaluated in the following manner.

The binding activity to TIM-3-expressing cell is evaluated by thebinding assay described in the above 1-(6a) and a surface plasmonresonance method using such as the Biacore system described in the above(6b). Furthermore, it can be measured by fluorescent antibody technique[Cancer Immunol. Immunother., 36, 373 (1993)] and the like.

Complement-Dependent Cytotoxicity (hereinafter, referred to as “CDCactivity”) or ADCC activity against an antigen positive cell line isevaluated by a known method [Cancer Immunol. Immunother., 36, 373(1993)].

4. Method of Controlling Effector Activity of Antibody

As a method for controlling an effector activity of the monoclonalantibody of the present invention, a method for controlling an amount offucose (hereinafter, referred to also as “core fucose”) which is boundin α-1,6 linkage to N-acetylglucosamine (GlcNAc) present in a reducingend of a complex type N-linked sugar chain which is bound to asparagine(Asn) at position 297 of an Fc region of an antibody (WO2005/035586,WO2002/31140, and WO00/61739), a method for controlling an effectoractivity of a monoclonal antibody by modifying amino acid group(s) of anFc region of the antibody, and the like are known. The effector activityof the anti-TIM-3 monoclonal antibody of the present invention can becontrolled by using any of the methods.

The “effector activity” means an antibody-dependent activity which isinduced via an Fc region of an antibody. As the effector activity, ADCCactivity, CDC activity, an antibody-dependent phagocytosis (ADPactivity) by phagocyte such as macrophages or dendritic cells, and thelike are known.

By controlling a content of core fucose of a complex type N-linked sugarchain of Fc of an antibody, an effector activity of the antibody can beincreased or decreased. According to a method for lowering a content offucose which is bound to a complex type N-linked sugar chain bound to Fcof the antibody, an antibody to which fucose is not bound can beobtained by the expression of an antibody using a CHO cell which isdeficient in a gene encoding α1,6-fucosyltransferase. The antibody towhich fucose is not bound has a high ADCC activity.

On the other hand, according to a method for increasing a content offucose which is bound to a complex type N-linked sugar chain bound to Fcof an antibody, an antibody to which fucose is bound can be obtained bythe expression of an antibody using a host cell into which a geneencoding α1,6-fucosyltransferase is introduced. The antibody to whichfucose is bound has a lower ADCC activity than the antibody to whichfucose is not bound.

Further, by modifying amino acid residue(s) in an Fc region of anantibody, the ADCC activity or CDC activity can be increased ordecreased. For example, the binding activity to an antibody can beincreased by using the amino acid sequence of the Fc region described inUS2007/0148165. Further, the ADCC activity or CDC activity can beincreased or decreased by modifying the amino acid as described in U.S.Pat. No. 6,737,056, or 7,297,775 or 7,317,091.

Furthermore, an antibody in which the effector activity is controlledcan be obtained by combining the above method within one antibody.

5. Method for Treating Disease Using the Anti-TIM-3 Monoclonal Antibodyor Antibody Fragment of the Present Invention

The monoclonal antibody or an antibody fragment thereof of the presentinvention can be used for treating a disease relating to a TIM-3positive cell.

The therapeutic agent comprising the antibody or antibody fragment ofthe present invention or derivatives thereof may be only the antibody orantibody fragment or derivatives thereof as an active ingredient, and ispreferably supplied as a pharmaceutical preparation produced by anappropriate method well known in the technical field of pharmaceutics,by mixing it with one or more pharmaceutically acceptable carriers.

Examples of a route of administration include oral administration andnon-oral administration, such as buccal, tracheal, rectal, subcutaneous,intramuscular or intravenous administration. Examples of the dosage formincludes sprays, capsules, tablets, powder, granules, syrups, emulsions,suppositories, injections, ointments, tapes and the like.

The pharmaceutical preparation suitable for oral administration includesemulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations such as emulsions and syrups can be produced using,as additives, water; sugars such as sucrose, sorbitol and fructose;glycols such as polyethylene glycol and propylene glycol; oils such assesame oil, olive oil and soybean oil; antiseptics such asp-hydroxybenzoic acid esters; flavors such as strawberry flavor andpeppermint; and the like.

Capsules, tablets, powders, granules and the like can be produced using,as additives, excipients such as lactose, glucose, sucrose and mannitol;disintegrating agents such as starch and sodium alginate; lubricantssuch as magnesium stearate and talc; binders such as polyvinyl alcohol,hydroxypropylcellulose and gelatin; surfactants such as fatty acidester; plasticizers such as glycerin; and the like.

The pharmaceutical preparation suitable for parenteral administrationincludes injections, suppositories, sprays and the like.

Injections can be prepared using a carrier such as a salt solution, aglucose solution or a mixture of both thereof.

Suppositories can be prepared using a carrier such as cacao butter,hydrogenated fat or carboxylic acid.

Sprays can be prepared using the antibody or antibody fragment as suchor using it together with a carrier which does not stimulate the buccalor airway mucous membrane of the patient and can facilitate absorptionof the compound by dispersing it as fine particles. The carrier includeslactose, glycerol and the like. It is possible to produce pharmaceuticalpreparations such as aerosols and dry powders.

In addition, the components exemplified as additives for oralpreparations can also be added to the parenteral preparations.

6. Method for Diagnosing Disease Using the Anti-TIM-3 MonoclonalAntibody or Antibody Fragment Thereof in the Present Invention

A disease relating to TIM-3 can be diagnosed by detecting or determiningTIM-3 or a cell expressing TIM-3 using the monoclonal antibody orantibody fragment of the present invention.

A diagnosis of cancer which is one of the diseases relating to TIM-3 canbe carried out by, for example, the following detection or measurementof TIM-3.

The diagnosis of cancer can be carried out by detecting TIM-3 expressionon the cell in a patient's body by an immunological method such as aflow cytometry.

An immunological method is a method in which an antibody amount or anantigen amount is detected or determined using a labeled antigen orantibody. Examples of the immunological method include radioactivesubstance-labeled immunoantibody method, enzyme immunoassay, fluorescentimmunoassay, luminescent immunoassay, Western blotting method,physico-chemical means and the like.

Examples of the radioactive substance-labeled immunoantibody methodinclude a method, in which the antibody or antibody fragment of thepresent invention is allowed to react with an antigen, a cell expressingan antigen or the like, then anti-immunoglobulin antibody subjected to aradioactive labeling or a binding fragment thereof is allowed to reacttherewith, followed by determination using a scintillation counter orthe like.

Examples of the enzyme immunoassay include a method, in which theantibody or antibody fragment of the present invention is allowed toreact with an antigen, a cell expressing an antigen or the like, then ananti-immunoglobulin antibody or an binding fragment thereof subjected toantibody labeling is allowed to react therewith and the colored pigmentis measured by a spectrophotometer, and, for example, sandwich ELISA maybe used.

As a label used in the enzyme immunoassay, any known enzyme label(Enzyme Immunoassay, published by Igaku Shoin, 1987) can be used asdescribed previously. Examples include alkaline phosphatase labeling,peroxidase labeling, luciferase labeling, biotin labeling and the like.

Sandwich ELISA is a method in which an antibody is bound to a solidphase, antigen to be detected or measured is trapped and anotherantibody is allowed to react with the trapped antigen. In the ELISA, twokinds of antibody which recognizes the antigen to be detected ormeasured or the antibody fragment thereof in which antigen recognizingsite is different are prepared and the first antibody or antibodyfragments is previously adsorbed on a plate (such as a 96-well plate)and the second antibody or antibody fragment is labeled with afluorescent substance such as FITC, an enzyme such as peroxidase, orbiotin.

The plate to which the above antibody is adsorbed is allowed to reactwith the cell separated from living body or disrupted cell suspensionthereof, tissue or disintegrated solution thereof, supernatant of cellculture, serum, pleural effusion, ascites, eye solution or the like,then allowed to react with a labeled monoclonal antibody or an antibodyfragment and a detection reaction corresponding to the labeled substanceis carried out. The antigen concentration in the sample to be tested canbe calculated from a calibration curve prepared by a stepwise dilutionof antigen of known concentration.

As antibody used for sandwich ELISA, any of polyclonal antibody andmonoclonal antibody may be used or antibody fragments such as Fab, Fab′and F(ab)₂ may be used. As a combination of 2 kinds of antibodies usedin sandwich ELISA, a combination of monoclonal antibodies or antibodyfragments recognizing different epitopes may be used or a combination ofpolyclonal antibody with monoclonal antibody or antibody fragments maybe used.

A fluorescent immunoassay includes a method described in the literatures[Monoclonal Antibodies—Principles and practice, Third Edition, AcademicPress (1996); Manual for Monoclonal Antibody Experiments, KodanshaScientific (1987)] and the like. As a label for the fluorescentimmunoassay, any of known fluorescent labels [Fluorescent Immunoassay,by Akira Kawao, Soft Science, (1983)] may be used. Examples of the labelinclude FITC, RITC and the like.

The luminescent immunoassay can be carried out using the methodsdescribed in the literature [Bioluminescence and Chemical Luminescence,Rinsho Kensa, 42, Hirokawa Shoten (1998)] and the like. As a label usedfor luminescent immunoassay, any of known luminescent labels can beincluded. Examples include acridinium ester, lophine or the like may beused.

Western blotting is a method in which an antigen or a cell expressing anantigen is fractionated by SDS (Sodium dodecyl sulfate)-PAGE[Antibodies—A Laboratory Manual (Cold Spring Harbor Laboratory, 1988)],the gel is blotted onto PVDF membrane or nitrocellulose membrane, themembrane is allowed to react with antigen-recognizing antibody orantibody fragment, further allowed to react with an anti-mouse IgGantibody or antibody fragment which is labeled with a fluorescentsubstance such as FITC, an enzyme label such as peroxidase, a biotinlabeling, or the like, and the label is visualized to confirm thereaction. An example thereof is described below.

Cells or tissues in which a polypeptide having the amino acid sequencerepresented by SEQ ID NO: 53 is expressed are dissolved in a solutionand, under reducing conditions, 0.1 to 30 μg as a protein amount perlane is electrophoresed by an SDS-PAGE method. The electrophoresedprotein is transferred onto a PVDF membrane and allowed to react withPBS containing 1 to 10% of BSA (hereinafter referred to as “BSA-PBS”) atroom temperature for 30 minutes for blocking.

Here, the monoclonal antibody of the present invention is allowed toreact therewith, washed with PBS containing 0.05 to 0.1% Tween 20(hereinafter referred to as “Tween-PBS”) and allowed to react with goatanti-mouse IgG labeled with peroxidase at room temperature for 2 hours.It is washed with Tween-PBS and a band to which the monoclonal antibodyis bound is detected using ECL Western Blotting Detection Reagents(manufactured by Amersham) or the like to thereby detect a polypeptidehaving the amino acid sequence represented by SEQ ID NO: 1. As anantibody used for the detection in Western blotting, an antibody whichcan be bound to a polypeptide having no three-dimensional structure of anatural type is used.

The physicochemical method is specifically carried out by reacting TIM-3as the antigen with the antibody or antibody fragment of the presentinvention to form an aggregate, and detecting this aggregate. Otherexamples of the physicochemical methods include a capillary method, aone-dimensional immunodiffusion method, an immunoturbidimetry, a lateximmunoturbidimetry [Handbook of Clinical Test Methods, Kanehara Shuppan,(1988)] and the like.

For example, in a latex immunodiffusion method, a carrier such aspolystyrene latex having a particle size of about of 0.1 to 1 μmsensitized with antibody or antigen may be used and when anantigen-antibody reaction is carried out using the corresponding antigenor antibody, scattered light in the reaction solution increases whiletransmitted light decreases. When such a change is detected asabsorbance or integral sphere turbidity, it is possible to measureantigen concentration, etc. in the sample to be tested.

In addition, for the detection of the cell expressing TIM-3, knownimmunological detection methods can be used, and an immunoprecipitationmethod, an immuno cell staining method, an immune tissue stainingmethod, a fluorescent antibody staining method and the like arepreferably used.

An immunoprecipitation method is a method in which a cell expressingTIM-3 is allowed to react with the monoclonal antibody or antibodyfragment of the present invention and then a carrier having specificbinding ability to immunoglobulin such as protein G-Sepharose is addedso that an antigen-antibody complex is precipitated. Also, the followingmethod can be carried out. The above-described antibody or antibodyfragment of the present invention is solid-phased on a 96-well plate forELISA and then blocked with BSA-PBS.

When the antibody is in a non-purified state such as a culturesupernatant of hybridoma cell, anti-mouse immunoglobulin or ratimmunoglobulin or protein A or Protein G or the like is previouslyadsorbed on a 96-well plate for ELISA and blocked with BSA-PBS and aculture supernatant of hybridoma cell is dispensed thereto for binding.After BSA-PBS is discarded and the residue is sufficiently washed withPBS, reaction is carried out with a dissolved solution of cells ortissues expressing TIM-3. An immune precipitate is extracted from thewell-washed plate with a sample buffer for SDS-PAGE and detected by theabove-described Western blotting.

An immune cell staining method or an immune tissue staining method are amethod where cells or tissues in which antigen is expressed are treated,if necessary, with a surfactant, methanol or the like to make anantibody easily permeate to the cells or tissues, then the monoclonalantibody of the present invention is allowed to react therewith, thenfurther allowed to react with an anti-immunoglobulin antibody or bindingfragment thereof subjected to fluorescent labeling such as FITC, enzymelabel such as peroxidase or biotin labeling and the label is visualizedand observed under a microscope.

In addition, cells can be detected by an immunofluorescent stainingmethod where cells are allowed to react with a fluorescence-labeledantibody and analyzed by a flow cytometer [MonoclonalAntibodies—Principles and practice, Third Edition, Academic Press(1996), Manual for Experiments of Monoclonal Antibodies, KodanshaScientific (1987)] in which cells are allowed to react with afluorescence-labeled antibody and analyzed by a flow cytometer.

Particularly, the monoclonal antibody or antibody fragment of thepresent invention which binds to amino acids sequences of anextracellular region of the TIM-3 or a three-dimensional structurethereof can detect a cell expressing the polypeptide maintaining anatural three-dimensional structure by a fluorescence antibody method.

In addition, in the case of using FMAT8100HTS system (manufactured byApplied Biosystems) and the like among fluorescent antibody stainingmethods, the antigen quantity or antibody quantity can be measuredwithout separating the formed antibody-antigen complex and the freeantibody or antigen which is not concerned in the formation of theantibody-antigen complex.

The present invention can provide a monoclonal antibody or an antibodyfragment of thereof, which binds to amino acids sequences of anextracellular region of TIM-3 or a three-dimensional structure andexpresses ADCC activity; a hybridoma which produces the antibody; a DNAwhich encodes the antibody; a vector which comprises the DNA; atransformant obtained by transforming the vector; a process forproducing an antibody or an antibody fragment thereof using thehybridoma or the transformant; and a therapeutic agent or a diagnosticagent comprising the antibody or the antibody fragment thereof as anactive ingredient.

Hereinafter, exemplary embodiments of the present invention will bedescribed specifically. However, the present invention is not limited tothe exemplary embodiments described below.

EXAMPLE Example 1 Molecular Cloning of Human TIM-3 cDNA andEstablishment of Stable Expression Cell

Human TIM-3 cDNA was amplified by PCR using ExTaq (manufactured byTakara Bio Inc.) and Human MTC panel (manufactured by Clontech) as atemplate. For primers, TIM-3 Fw2 (SEQ ID NO: 31) and TIM-3 Re2 (SEQ IDNO: 32) were used. The obtained PCR product was inserted into pGEM-TEasy vector (manufactured by Promega Corp.), and the plasmid wasintroduced into TOP10 One shot competent cells (manufactured byInvitrogen) to amplify. The plasmid DNA was extracted by the miniprepmethod.

By analyzing the sequence of purified DNA using primers TIM-3 Fw andTIM-3 Re2, the clone having the same sequence as the coding region ofGenBank accession number NM_032782 was selected. A human TIM-3retrovirus (hTIM-3/pMCs-IG) was constructed by recombination of theinsert of the selected clone into pMCs-IRES-GFP vector (manufactured byCell Biolabs Inc.) using Retrovirus Constructive System Ampho(manufactured by Takara Bio Inc.).

The human TIM-3 retrovirus was infected into Jurkat cell (ATCC accessionNO: CRL-2063), EoL-1 cell (RIKEN CELL BANK NO: RCB0641) and L929 cell(manufactured by Sigma-Aldrich Co., LLC.) which were confirmed not toendogenously express TIM-3 was confirmed by flow cytometry analysis.After several passages, TIM-3 positive cells were collected by FACSAria(manufactured by BD Biosciences). Human TIM-3 stable expression cellline was established by recollecting the TIM-3 positive cells byFACSAria after several passages.

Example 2 Preparation of Expression Vector of Fusion Protein of SolubleExtracellular Human TIM-3-Human Fc

The cDNA encoding the extracellular region of human TIM-3 was amplifiedby PCR using ExTaq (manufactured by Takara Bio Inc.) and hTIM-3/pMCs-Igconstructed in Example 1 as a template. For primers, pMCs-Fw (SEQ ID NO:33) and TIM3ED-FcReXba 2 (SEQ ID NO: 34) were used.

The amplified PCR product was inserted into pTracer-CMV-FLAG-human Fcvetctor [modified vector in which FLAG and Fc domain of human IgG1 wereinserted into pTracer-CMV (manufactured by Invitrogen) vector betweenXbaI site and ApaI site]. After the plasmid was introduced into TOP10One shot competent cells (manufactured by Invitrogen) to amplify, theplasmid DNA (sTIM-3-FLAG-Fc/pTracerCMV) was extracted by miniprepmethod.

By the analysis of the DNA sequence using T7 (SEQ ID NO: 35) and hTIM-3Fw1 (SEQ ID NO: 36) as a primer, the purified sTIM-3-FLAG-Fc/pTracerCMVwas confirmed to have the same nucleotide sequence as the correspondingregion in GenBank accession number NM_032782. The nucleotide sequence(from EcoRI recognition site to ApaI recognition site) was identical tothe nucleotide sequence represented by SEQ ID NO: 37.

Example 3 Preparation of Expression Vector of Soluble ExtracellularHuman TIM-3

The cDNA encoding the extracellular region of human TIM-3 was amplifiedby PCR using ExTaq (manufactured by Takara Bio Inc.) andsTIM-3-FLAG-Fc/pTracerCMV established in Example 2 as a template. Asprimers, TIM-3 Fw2 (SEQ ID NO: 31) and TIM3ED-FLAG4aa (SEQ ID NO: 39)were used.

Next, FLAG epitope was ligated by PCR method using ExTaq (manufacturedby Takara Bio Inc.), TIM-3 Fw2 (SEQ ID NO: 31) and C-FLAG-NotR2 (SEQ IDNO: 40) as primers and the obtained PCR product as a template. The PCRproduct was inserted into pGEM-T Easy vector (manufactured by PromegaCorp.). After the plasmid was introduced into TOP10 One shot competentcells (manufactured by Invitrogen) to amplify the plasmid DNA(sTIM-3-FLAG/pEF6Myc_HisC) was extracted by the miniprep method.

By the DNA sequence analysis using T7 (SEQ ID NO: 35) and BGH-R (SEQ IDNO: 41) as a primer, the purified sTIM-3-FLAG-Fc/pEF6 Myc_HisC wasconfirmed to have the same nucleotide sequence as the correspondingregion in GenBank accession number NM_032782.

Example 4 Preparation of the Fusion Protein of Soluble ExtracellularHuman TIM-3-Human Fc and Soluble Extracellular Human TIM-3

Each of sTIM-3-FLAG-Fc/pTracerCMV plasmid DNA and sTIM-3-FLAG-Fc/pEF6Myc_HisC plasmid DNA obtained in Example 2 and Example 3, respectively,was introduced into HEK293F cells (manufactured by Invitrogen) and wastransiently expressed. Six days after, each of the cell supernatant wasrecovered and used for protein purification.

The culture supernatant containing the fusion protein of solubleextracellular human TIM-3-human Fc or soluble extracellular human TIM-3was recovered by centrifugation six days after transfection. The fusionproteins were purified by an anti-FLAG column prepared using Anti-FLAGM2 Agarose Affinity gel (manufactured by Sigma-Aldrich Co., LLC.) and byFLAG peptides (manufactured by Sigma-Aldrich Co., LLC.) according to themanufacturer's protocols.

The eluted samples were fractionated and then each fraction was analyzedby SDS-PAGE under reduced conditions, and then silver staining andWestern blotting were carried out. For Western blotting, an anti-FLAG M2antibody (manufactured by Sigma-Aldrich Co., LLC.) and an alkalinephosphatase labeled rabbit anti-mouse immunoglobulin antibody was used(manufactured by Dako). The fraction containing the target protein wasconcentrated using Amicon Ultra 4 10K (manufactured by Millipore) andwas applied for gel filtration chromatography using Superdex 200 gpcolumn (manufactured by GE Healthcare).

After fractionation, each fraction was applied to SDS-PAGE under reducedcondition, silver stained and then Western blotted. The fraction inwhich the target protein was found was concentrated and washed with 0.5ml of PBS. The soluble extracellular human TIM-3-human Fc fusion proteinor soluble extracellular human TIM-3 was collected after filteringthrough the MILLEX-GV sterilizing filter (manufactured by Millipore)having a pore diameter of 0.22 μm. The purity of the protein wasmeasured by Limulus ES-II kit Wako (manufactured by Wako Pure ChemicalIndustries, Ltd.) which is a kit to detect endotoxin and confirmed thatthe fraction was sufficiently purified.

Example 5 Preparation of Human Antibody Expressing Mouse

The mice used for immunization has a genetic background in which both ofendogenous Ig heavy chain locus and κ light chain locus werehomozygously disrupted and maintained a chromosome 14 fragment (SC20)comprising a human heavy chain transchromosome, and a human Igκ (chaintransgene (KCo5) at the same time. This mouse strain was established bycross breeding mice strain A which had human Ig heavy chain loci andmice strain B which maintained human Igκ chain transgene.

Mouse strain A is a homozygote in which both of the endogenous Ig heavychain locus and κ light chain locus are homozygously disrupted, andmaintains a chromosome 14 fragment (SC20) which can be transmitted tothe progeny and for example, described in Tomizuka et al. (Tomizuka, K.et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97:722-727).

Mouse Strain B is a mouse strain (transgenic mouse) in which both of theendogenous Ig heavy chain locus and κ light chain locus are homozygouslydisrupted, and maintains a human Igκ (light chain transgene (KCo5), andis, for example, described in Fishwild et al. (1996) [NatureBiotechnology 14:845-851].

The animal individual was obtained by cross-breeding male mouse ofstrain A and female mouse of strain B, or female mouse of strain A andmale mouse of strain B and was a mouse in which human Ig heavy chain andIgκ (light chain could be detected at the same time in their serum[Ishida & Lonberg, IBC's 11th Antibody Engineering, Abstract 2000] wasfurther used for immunological experiments. For your information, thehuman antibody expressing mouse (hereinafter referred to as “KM mouse”)can be obtained from Kyowa Hakko Kirin by establishing a license.

Example 6 Preparation of Anti-Human TIM-3 Monoclonal Antibody

The preparation of monoclonal antibody in the present Example wascarried out according to the general methods, such as published inMonoclonal Antibody Experiment Manual, (Yasuhigashi Tamie et al.,Kodansha, 1991). Either TIM-3 expressing L929 cell prepared in Example 1or the soluble extracellular human TIM-3-human Fc fusion proteinprepared in Example 4 was used as a TIM-3 immunogen. The immunizedanimal was KM mouse described above.

The TIM-3 expressing L929 cell was intraperitoneally injected into miceat a dose of 1×10⁷ cells/animal. After the first immunization, the samecell was further administered for another three times or morethereafter. Three days before obtaining the spleen, the solubleextracellular human TIM-3-human Fc fusion protein prepared in Example 4was administered from caudal vein at 20 μg/mouse. The spleen wassurgically removed from the immunized mouse, put into 4 ml of PBS andmashed on a mesh (cell strainer: manufactured by Falcon) using a syringepiston.

The cells were precipitated by centrifuging the cell suspension whichwas passed through the mesh, and the cells were resuspended in 1 ml ofRed Blood Cell Lysis Buffer (manufactured by Sigma-Aldrich Co., LLC.).After incubating for 5 min at room temperature, 10 ml of serum free DMEMmedium (manufactured by Invitrogen) (hereinafter referred to as “serumfree DMEM medium”) containing 50 unit/ml penicillin and 50 μg/mlstreptomycin was added, and the cells were precipitated bycentrifugation.

The cell pellets were resuspended in serum free DMEM medium. On theother hand, myeloma cell line SP2/0 (ATCC NO: CRL-1581) was cultured inDMEM medium containing 10% FBS, 50 unit/ml penicillin and 50 μg/mlstreptomycin (hereinafter referred to as “DMEM medium containing serum”)and incubated at 37° C. under 5% CO₂ so as not exceed the cell number of1×10⁶ cells/ml.

The SP2/0 cells were washed with 10 ml of serum free DMEM medium andsuspended in serum free DMEM medium as in the case of the spleen derivedcells. These suspension of spleen derived cells and suspension ofmyeloma cell were mixed at a ratio of 5:1, centrifuged, and thesupernatant was completely removed. To fuse the cells, 1 ml of 50% (w/v)of polyethylene glycol (manufactured by Boehringer Mannheim Corp.) wasslowly added while stirring the pellet with the pipette tip, and then 1ml of serum free DMEM medium preheated at 37° C. was added. Slowly, 5 mland 10 ml of serum free DMEM was added followed by 5 min incubation at37° C. under 5% CO₂.

After the centrifugation and removal of supernatant, the fused cellswere resuspended in 50 ml of DMEM medium containing 10% FBS(manufactured by Invitrogen), penicillin-streptomycin-glutamine(manufactured by Invitrogen, 100-fold dilution of Catalog No.10378-016), IL-6 (5 ng/ml), and 2-mercaptoethanol (manufactured byInvitrogen, 1000-fold dilution of Catalog No. 21985-023) (hereinafterreferred to as “DMEM medium containing IL-6”) and incubated at 37° C.under 5% CO₂.

The next day, cells were harvested by pipetting, centrifuged and thepellet was resuspended in 10 ml of DMEM medium containing IL-6. On thenext day, Hypoxanthine-aminopterin-thymidine (hereinafter referred to as“HAT”, manufactured by Sigma-Aldrich Co., LLC.) was added. Afterincubating for about 7 to 10 days, the culture supernatant was collectedfor hybridoma screening.

Example 7 Preparation of Anti-Human TIM-3 Mouse Monoclonal Antibody

The preparation of monoclonal antibody in the present Example wascarried out according to the general methods published in Introductionto Monoclonal Antibody Experiment (Yasuhigashi Tamie et al., Kodansha,1991). Either TIM-3 expressing L929 cell prepared in Example 1 orsoluble extracellular human TIM-3 prepared in Example 4 was used as aTIM-3 immunogen. The immunized animal was Balb/c mouse (purchased fromNihon Charles River).

First, a Balb/c mouse was immunized by injecting TIM-3 expressing L929cells intraperitoneally at a dose of 1×10⁷ cells/animal. After the firstimmunization, the same cell was further administered for another threetimes or more thereafter. Three days before extracting the spleen, thesoluble extracellular human TIM-3 protein prepared in Example 4 wasadministered from caudal vein at 20 μg/mouse. The spleen was surgicallyremoved from the immunized mouse, put into 4 ml of PBS and mashed on amesh (cell strainer: manufactured by Falcon) using a syringe piston.

The cells were precipitated by centrifuging the cell suspension whichwas passed through the mesh, and the cells were resuspended in 1 ml ofRed Blood Cell Lysis Buffer (manufactured by Sigma-Aldrich Co., LLC.).After incubating for 5 min at room temperature, 10 ml of serum free DMEMmedium was added, and then cells were precipitated by centrifugation.The pellets were resuspended in serum free DMEM medium. On the otherhand, myeloma cell line SP2/0 (ATCC NO: CRL-1581) were cultured in DMEMmedium by incubating at 37° C. under 5% CO₂ to the cell number of 1×10⁶cells/ml or less.

Similar to the spleen derived cells, the SP2/0 cells were washed with 10ml of serum free DMEM medium and suspended in serum free DMEM medium.The suspension of spleen cell and the suspension of myeloma cell weremixed at a ratio of 5:1. After centrifugation, the supernatant wasremoved. To this pellet, 1 ml of 50% (w/v) of polyethylene glycol(manufactured by Boehringer Mannheim Corp.) was slowly added whilestirring the pellet with the pipette tip, and then 1 ml of serum freeDMEM medium preheated to 37° C. was added.

To this suspension, 5 ml of DMEM medium was slowly added. After furtheradding 10 ml of serum free DMEM, the suspension was incubated for 5 minat 37° C. under 5% CO₂. After the suspension was centrifuged, the cellpellets were resuspended in 50 ml of DMEM medium containing IL-6 andcultured at 37° C. under 5% CO₂. After incubation for one day, cellswere collected by pipetting. After centrifugation, the pellet wasresuspended in 10 ml of DMEM medium containing IL-6. On the next day,HAT medium (manufactured by Sigma-Aldrich Co., LLC.) was added. Afterincubation for about 7 to 10 days, the culture supernatant was recoveredand used for hybridoma screening.

Example 8 Screening of Hybridoma Producing a Human or Mouse MonoclonalAntibody which Binds to Human TIM-3

The hybridoma was screened using the cell supernatant prepared inExample 6 and Example 7. The method to be used was a flow cytometrymethod using human TIM-3 expressing cell lines. First, the human TIM-3expressing Jurkat cell or EoL-1 cell prepared in Example 1 were washedwith staining medium (PBS containing 2% FBS and 0.05% sodium azide), andthen resuspended in 1 ml of staining medium to give a cell density of1×10⁶ cells/ml. The cell suspension was dispensed at 10 μl/well into a96-well culture plate.

Then, 50 μl of the hybridoma supernatant was added and incubated for 30min at 4° C. After the cells were washed twice with the staining medium,50 μl of labeled antibody which was diluted 200-fold with stainingmedium was added to each well and incubated for 30 min at 4° C. As forlabeled antibodies, Goat F(ab′)₂ Anti-Human IgG (γ chain specific)-R-PE(manufactured by SouthernBiotech) were used for the human monoclonalantibody and Goat F(ab′)₂ Anti-Mouse IgG(H+L)R-PE (manufactured bySouthernBiotech) was used for the mouse monoclonal antibody.

After washing twice with staining medium, the cells were analysed usingFACSCalibur (manufactured by BD Biosciences) as a primary screening.After positive clones were picked and expanded, the hybridoma cells wereclone-sorted by FACSAria (manufactured by BD Biosciences) and culturedfor seven days in DMEM medium containing IL-6 which further containsHypoxanthine-thymidine (hereinafter referred to as “HT”, manufactured bySigma-Aldrich Co., LLC.).

Harvested supernatants were screened as the same as in the primaryscreening method for cloning of hybridomas expressing anti-human TIM-3human monoclonal antibody and hybridomas expressing anti-human TIM-3mouse monoclonal antibody and selected hybridoma clone was used forpurification of monoclonal antibody.

Example 9 Purification of Hybridoma Derived Monoclonal Antibody

An anti-TIM-3 human or mouse monoclonal antibody was purified from theanti-human TIM-3 human or mouse monoclonal antibody expressing hybridomaprepared in Example 8. Briefly, hybridoma supernatant containing a highconcentration of the anti-TIM-3 antibody was prepared using a CELLineantibody production system (manufactured by BD Biosciences) and theanti-human TIM-3 monoclonal antibody was purified from the supernatant.First, to adapt the cloned hybridoma to the serum free medium, thehybridomas were cultured for several days in a medium mixing DMEM mediumcontaining HT and IL-6 and BD Cell MAb serum free medium (manufacturedby BD Biosciences) at a ratio of 1:1, and then cultured for several daysin a medium having a mixing ratio of 1:2.

Next, the hybridomas were cultured in a BD Cell MAb serum free medium(manufactured by BD Biosciences) and adapted hybridomas to the serumfree medium. The hybridoma cells adapted to the serum free medium werecultured in CL-1000 flasks according to the manufacturer's instructionsand the supernatant of the hybridoma containing high concentration ofthe anti-TIM-3 antibody was collected from the flasks. The antibody fromthe supernatant of the hybridoma was purified in accordance with thestandard procedures using Protein A.

Specifically, MabSelect (manufactured by GE Healthcare) were packed inan open column and the supernatant diluted 2-fold by PBS was loaded andthen eluted with 0.1 mol/L Glycine-HCl (pH 2.7), followed byconcentration with Amicon Ultra (manufactured by Millipore). Next, byusing a NAP-5 column (manufactured by GE Healthcare), the obtainedsolution was equilibrated with PBS buffer, and then sterilized byfiltration to obtain 4 clones of the anti-human TIM-3 human monoclonalantibodies (antibody 512, antibody 644, antibody 4545 and antibody 4177)and one clone (antibody 8213) of the anti-human TIM-3 mouse monoclonalantibody.

Example 10 Identification of Isotype of Anti-TIM-3 Human MonoclonalAntibody

Each of the isotype of anti-TIM-3 human monoclonal antibodies which wasprepared in Example 9 was determined by a solid-phase ELISA and flowcytometry.

Specifically, as for the solid-phase ELISA, soluble extracellular humanTIM-3 obtained by Example 4 was diluted with a Carbonate-BiacarbonateBuffer (manufactured by Sigma-Aldrich Co., LLC.) to give a concentrationof 1 μg/ml and dispensed at 50 μl/well into a 96-well culture plate(Maxisorp, manufactured by Nunc). The soluble extracellular human TIM-3was adsorbed to the microplate by incubating overnight at 4° C.

After the supernatant was discarded, SuperBlock Blocking Buffer in TBS(manufactured by PIERCE) was added to the plate and then incubated for10 min at room temperature and 50 μl of the culture supernatant of thehybridoma was added and then incubated for 30 min at room temperature.After washing each well with Tris-buffered saline containing 0.1%Tween20 (TBS-T), 50 μl of each of HRP-labeled mouse anti-human IgG1,anti-human IgG2, anti-human IgG3 or anti-human IgG4 antibodies, eachantibody was diluted 2000, 2000, 2000 and 4000-fold with TBS-Tcontaining 10% SuperBlock Blocking Buffer, respectively; manufactured bySouthernBiotech), were added and incubated for 30 minutes at roomtemperature.

After washing each well with TBS-T, 50 μl of substrate buffer (TMB,manufactured by DAKO) was added and then incubated for 20 min at roomtemperature. The reaction was stopped by adding 50 μl of 0.5 mol/Lsulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.). Theabsorbance at wavelength of 450 nm (reference wavelength: 570 nm) wasmeasured on a microplate reader (VersaMax, manufactured by MolecularDevices, LLC.).

In addition, in flow cytometry method, the Jurkat cell which expressedhuman TIM-3 prepared in Example 1 was washed with staining medium, andthen 50 μl of the supernatant of the hybridoma was added and incubatedfor 30 min at 4° C. After washing, 50 μl of each of Mouse anti-HumanIgG1-PE, Mouse anti-Human IgG2-PE, Mouse anti-Human IgG3 (Hinge)-PE andMouse anti-Human IgG4(Fc)-PE (manufactured by SouthernBiotech) which wasdiluted 100-fold with staining medium was added, allowed to stand stillfor 30 min at 4° C. and then analyzed using FACSCalibur flow cytometer(manufactured by BD Biosciences).

Based on the result, the isotypes of antibody 512, antibody 644, andantibody 4545 were IgG1, IgG4 and IgG2, respectively.

Example 11 Identification of the Isotype of the Mouse MonoclonalAntibody for Human TIM-3

The isotype of the anti-human TIM-3 mouse monoclonal antibody preparedin Example 9 was determined using Iso Strip Mouse Monoclonal AntibodyIsotyping Kit (manufactured by Roche-diagnostics). By using thesupernatant of the hybridoma containing the anti-TIM-3 antibody preparedin Example 8, the isotype was determined according to the manufacturer'sinstructions. As a result, the isotype of antibody 8213 was determinedas IgG2b.

Example 12 Isolation of Anti-TIM-3 Monoclonal Antibody Gene

The gene of anti TIM-3 human or mouse monoclonal antibody which wasprepared in Example 9 was isolated from the hybridoma which producedanti-human TIM-3 human or mouse monoclonal antibody prepared in Example9. Total RNA was extracted from the cloned hybridoma using High Pure RNAIsolation Kit (manufactured by Roche-Diagnostics), according to themanufacturer's instructions. The cloning of the cDNA encoding thevariable region was carried out using SMART RACE cDNA amplification Kit(manufactured by Clontech) in accordance with the instructions attachedthereto.

The heavy chain (VH) of the human monoclonal antibody was amplified byPCR using UPM (SMART RACE cDNA amplification Kit; manufactured byClontech) and primer hh-6 (SEQ ID NO: 44). Using part of the reactionproduct as a template, PCR was carried out using NUP (SMART RACE cDNAamplification Kit; manufactured by Clontech) and primer hh-3 (SEQ ID NO:45). The cloning of the PCR product was carried out using Zero BluntTOPO PCR Cloning Kit (manufactured by Invitrogen). The nucleotidesequence was analyzed by universal primers of the vector (e.g., T7 orM13R primer) and the amino acid sequence was deduced from the nucleotidesequence.

As a result, the nucleotide sequence and the amino acid sequence of VHof antibody 512 were represented by SEQ ID NO: 54, was and SEQ ID NO:55, respectively, the nucleotide sequence and the amino acid sequence ofVH of antibody 644 were represented by SEQ ID NO: 58 and SEQ ID NO: 59,respectively, the nucleotide sequence and the amino acid sequence of VHof antibody 4545 were represented by SEQ ID NO: 7 and SEQ ID NO: 8,respectively, and the nucleotide sequence and the amino acid sequence ofVH of antibody 4177 were represented by SEQ ID NO: 17 and SEQ ID NO: 18,respectively. The protein sequences of SEQ ID NOS: 8 and 18 are shown tocontain the signal sequence which consists of the first 19 amino acidresidues. The mature protein sequence of the proteins of SEQ ID NOS: 8and 18 are shown as SEQ ID NO: 116 and 118, respectively. Each of theobtained variable regions obtained and the constant region of human IgG1were inserted and ligated into the expression vector, N5KG1(manufactured by Biogen IDEC Inc.).

Human monoclonal antibody light chain (VL) was amplified by PCR usingUPM (SMART RACE cDNA amplification Kit; manufactured by Clontech) andprimer hk-2 (SEQ ID NO: 46). Using part of the reaction product as atemplate, PCR was carried out using NUP (SMART RACE cDNA amplificationKit; manufactured by Clontech) and primer hk-6 (SEQ ID NO: 47). The PCRproduct was cloned using Zero Blunt TOPO PCR Cloning Kit (manufacturedby Invitrogen). The nucleotide sequence was analyzed by universalprimers of the vector (e.g., T7 or M13R) and the amino acid sequence wasdeduced from the nucleotide sequence.

As a result, the nucleotide sequence and the amino acid sequence of VLof antibody 512 were represented by SEQ ID NO: 56 and SEQ ID NO: 57,respectively, the nucleotide sequence and the amino acid sequence of VLof antibody 644 were represented by SEQ ID NO: 60 and SEQ ID NO: 61,respectively, the nucleotide sequence and the amino acid sequence of VLof antibody 4545 were represented by SEQ ID NO: 9 and SEQ ID NO: 10,respectively, and the nucleotide sequence and the amino acid sequence ofVL of antibody 4177 were represented by SEQ ID NO: 19 and SEQ ID NO: 20,respectively. The protein sequences of SEQ ID NOS: 10 and 20 are shownto contain the signal sequence which consists of the first 20 amino acidresidues. The mature protein sequence of the proteins of SEQ ID NOS: 10and 20 are shown as SEQ ID NO: 117 and 119, respectively. Each of theobtained variable regions and constant region of κ chain were insertedand ligated into the expression vector, N5KG1 (manufactured by BiogenIDEC Inc.).

The heavy chain (VH) of mouse monoclonal antibody was amplified by PCRusing UPM (SMART RACE cDNA amplification Kit; manufactured by Clontech)and a primer, mH_Rv1 (SEQ ID NO: 48). Using part of the reaction producte as template, PCR was carried out using NUP (SMART RACE cDNAamplification Kit; manufactured by Clontech) and primer mH_Rb2 (SEQ IDNO: 49).

The cloning of the PCR product was carried out using Zero Blunt TOPO PCRCloning Kit (manufactured by Invitrogen). The nucleotide sequence wasanalyzed by universal primers of the vector (e.g., T7 or M13R) and theamino acid sequence was deduced from the nucleotide sequence.

As a result, the nucleotide sequence and the amino acid sequence of VHof antibody 8213 were represented by SEQ ID NO: 27 and SEQ ID NO: 28,respectively. The obtained variable region and the constant region ofmouse IgG2a were ligated and inserted into the expression vector, N5KG1(manufactured by Biogen IDEC Inc.).

The light chain (VL) of the mouse monoclonal antibody was amplified byPCR using UPM (SMART RACE cDNA amplification Kit; manufactured byClontech) and primer mK_Rv1 (SEQ ID NO: 50). Additionally, part of thereaction product was used as a template, and PCR was carried out usingNUP (SMART RACE cDNA amplification Kit; manufactured by Clontech) andprimer mK_Rv2 (SEQ ID NO: 51). The PCR product was cloned using ZeroBlunt TOPO PCR Cloning Kit (manufactured by Invitrogen). Nucleotidesequences were analyzed by universal primers of the vector (e.g., T7 orM13R) and the amino acid sequence was deduced from the nucleotidesequence.

As a result, the nucleotide sequence and the amino acid sequence of theVL of antibody 8213 were represented by SEQ ID NO: 29 and SEQ ID NO: 30,respectively.

The variable region obtained and the constant region of κ chain wereligated and inserted into the above mentioned expression vector encodingVH of antibody 8213.

In addition, in order to obtain anti-dinitrophenyl (DNP) human IgG1antibody as a negative control, the nucleotide sequence of the heavychain (VH) of the antibody (SEQ ID NO: 62), the nucleotide sequence ofthe light chain of the antibody (SEQ ID NO: 64), and the constant regionof human IgG1 and the κ chain were inserted into expression vector N5KG1(manufactured by Biogen IDEC Inc.).

Example 13 Purification of Recombinant Anti-TIM-3 Human MonoclonalDefucose Antibody

A cell expressing a recombinant antibody was produced by introducing therecombinant antibody expression vector prepared in Example 12 into thehost cell. As the host cell for expression, FUT8^(−/−) CHO cells wereused in accordance with a published report (Clin Cancer Res 2006: 12(9)Lida et al.). Briefly, the expression vectors for each of 512, 644, 4545and 4177 prepared in Example 12 were introduced into FUT8^(−/−) CHO cellusing FreeStyle™ MAX Reagent (manufactured by Invitrogen). TheFUT8^(−/−) CHO cells were cultured on a shaker at 37° C. under 5% CO₂.The culture supernatant was recovered after about five days.

The recovered supernatant was purified using Protein A (manufactured byGE Healthcare) and further using 0.8×40 cm column (manufactured byBioRad) and the like, depending on the sample amount. Antibodies wereaffinity purified using PBS as the binding buffer and 0.02 mol/L sodiumcitrate (pH 2.7, 50 mmol/L NaCl) buffer as the elution buffer. Theelution fraction was added with 0.2 mol/L of sodium phosphate buffer (pH7.0).

The prepared antibody solution was equilibrated with PBS using NAP-25column (manufactured by GE Healthcare) and concentrated with AmiconUltra (10000 cut, manufactured by Millipore). After sterilization byfiltration, the recombinant anti-TIM-3 human monoclonal defucoseantibodies (antibody 512, antibody 644, antibody 4545 and antibody 4177)were obtained. The purity of the protein was determined by Limulus ES-IIkit Wako (manufactured by Wako Pure Chemical Industries, Ltd.) and foundthat these antibodies were sufficiently purified.

The vector for expressing anti-DNP prepared in Example 12 was introducedinto CHO-DG44 cells. The CHO-DG44 cells were cultured on a shaker at 37°C. under 5% CO₂. The cultured supernatant was recovered followingincubation for 5 days. The collected supernatant was purified usingProtein A (manufactured by GE Healthcare) and further using 0.8×40 cmcolumn (manufactured by BioRad) and the like, depending on the sampleamount. The affinity purification of the antibodies was carried outusing PBS as the binding buffer and 0.02 mol/L of sodium citrate (pH2.7, 50 mmol/L NaCl) buffer as the elution buffer. The elution fractionwas added with 0.2 mol/L of sodium phosphate buffer (pH 7.0). Theprepared antibody solution was equilibrated with PBS and concentratedusing NAP-25 column (manufactured by GE Healthcare) with Amicon Ultra(10000 cut, manufactured by Millipore) respectively. After sterilizationby filtration, the recombinant anti-DNP antibody was obtained. Thepurity of the protein was determined using a Limulus ES_II kit Wako(manufactured by Wako Pure Chemical Industries, Ltd.) and found that theantibody was sufficiently purified.

Example 14 Purification of Recombinant Anti-TIM-3 Mouse MonoclonalAntibody

The vector for expressing antibody 8213 prepared in Example 12 wasintroduced into HEK293F cells (manufactured by Invitrogen) using 293fectin (manufactured by Invitrogen). HEK293F cells were incubated on ashaker at 37° C. under 5% CO₂. The cultured supernatant was collectedafter incubation for five days. The collected supernatant was purifiedusing Protein A (manufactured by GE Healthcare) and further using 0.8×40cm column (manufactured by BioRad) and the like, depending on the sampleamount. The affinity purification of the antibody was carried out usingPBS as a binding buffer and 0.02 mol/L sodium citrate buffer (pH 2.7, 50mmol/L NaCl) as an elution buffer.

To the elution fraction, 0.2 mol/L of sodium phosphate buffer (pH 7.0)was added. The prepared antibody solution was equilibrated with PBSusing NAP-25 column (manufactured by GE Healthcare) and concentratedwith Amicon Ultra (10000 cut, manufactured by Millipore). Aftersterilization by filtration, the recombinant anti-TIM-3 mouse monoclonalantibody (8213 antibody) was obtained. The purity of the protein wasdetermined using a Limulus ES_II kit Wako (manufactured by Wako PureChemical Industries, Ltd.) and found that the antibody was sufficientlypurified.

Example 15 Fluorescent Labeling of Purified Monoclonal Antibody

The fluorescent labeling of the hybridoma derived monoclonal antibodyand recombinant monoclonal antibody was carried out by using Alexa Fluor647 Monoclonal Antibody Labeling Kit (manufactured by Invitrogen)according to the manufacturer's instructions. After the labeling, eachantibody was confirmed to be positively labeled with Alexa Fluor 647(hereinafter referred to as “Alexa-647”) by FACSCalibur (manufactured byBD Biosciences) analysis for TIM-3 expressing cells.

Example 16 Classification of Epitopes Using Competition Test-1

The relation of epitopes between antibodies prepared in Example 13 andExample 14, and commercially available anti-TIM-3 antibody 344823 (Clone344823, manufactured by R&D Systems) was analyzed by a competition test.In brief, the test antibody was allowed to interact with the TIM-3expression cell prepared in Example 1, and then whether anotheranti-TIM-3 antibody could further bind to TIM-3 expressing cells wasevaluated by flow cytometry method.

In the first step, whether or not the competition between each of themonoclonal antibodies prepared in Example 13 and Example 14 (antibody512, antibody 644, antibody 4177 and antibody 8213) and antibody 34823were examined. First, human TIM-3 expressing EoL-1 cells and the parentcell, EoL-1 cells were washed with staining medium and then allowed toreact with each of the purified subjective monoclonal antibodies(antibody 512, antibody 644, antibody 4177 and antibody 8213) (4° C., 30min). The final concentration was 10 μg/ml. Next, the PE labeledantibody 344823 (Clone 344823, manufactured by R&D Systems) was addedand allowed to react (4° C., 30 min). After washing with stainingmedium, 7-ADD (manufactured by BD Biosciences) was added and analyzedusing FACSCalibur (manufactured by BD Biosciences).

As a result, the goat derived anti-human TIM-3 polyclonal antibody(manufactured by R&D Systems) used as a positive control and antibody644 almost completely blocked the binding of PE-labeled antibody 344823to TIM-3 expressing cell. However, antibody 512, 4545 and 8213 did notinhibit the binding of PE-labeled antibody 344823 to TIM-3 expressingcell. Antibody 4177 inhibited the binding of PE-labeled antibody 344823to TIM-3 expressing cells, but this inhibition was weaker than that ofantibody 644. Accordingly, antibody 644 competed with antibody 344823,but antibodies 512, 4545 and 8213 did not compete with antibody 344823.It was found that the antibody 4177 and antibody 344823 competed witheach other, but they competed partially.

In the second step, among the anti-TIM-3 monoclonal antibodies tested,antibodies 512, 4545 and 8213 which did not interact with antibody344823 were examined whether or not they competed. The experiment wascarried out in the same manner as the first step except for usingAlexa-647 labeled antibody 512 or Alexa-647 labeled antibody 8213prepared in Example 15 as a labeled antibody. As a result, antibody 4545and antibody 8213 did not inhibit the binding of Alexa-647 labeledantibody 512 to the TIM-3 expressing cells.

However, antibody 4545 inhibited the binding of Alexa-647 labeledantibody 8213 to TIM-3 expressing cells. Therefore, it was found thatantibody 4545 and antibody 8213 competed with each other, but these twoantibodies did not compete with antibody 512. This result suggested thatantibody 512 recognized different epitope from antibody 344823 andantibody 8213.

In the third step, a competitive assay with antibody 512 and antibody8213 was carried out on antibody 4177 which was shown to partiallycompete with antibody 344823 in the first step. The experiment wasperformed in the same manner as described in second step. As a result,antibody 4177 did not inhibit the binding of the Alexa-647 labeledantibody 512 to the TIM-3 expressing cells, but inhibited the binding ofthe Alexa-647 labeled antibody 8213 to the TIM-3 expressing cells.Therefore, it was found that antibody 4177 did not compete with antibody512, but only with antibody 8213.

Based on these results, among the 5 antibodies prepared in Example 13and Example 14, antibody 644 recognized an epitope which was near theepitope to which antibody 344823 recognized; antibody 512 recognized anepitope different from of the epitope to which antibody 344823 and otherfour antibodies (antibody 644, antibody 4545, antibody 4177 and antibody8213) recognized.

It was suggested that antibody 8213 recognized epitope different from ofthe epitope to which antibodies 344823 or 512 recognized. It wassuggested that antibody 4545 recognized an epitope which was near theepitope to which antibody 8213 recognized. However, antibody 4177recognized the epitope which was near the epitope to which antibody 8213recognized, and also weakly recognized an epitope which was near theepitope to which antibody 344823 recognized.

Example 17 Antibody Dependent Cellular Cytotoxicity Test of RecombinantAnti-TIM-3 Human Monoclonal Defucose Antibody

Regarding the cellular cytotoxic activity via antibody, the ADCCactivity against the target cell was measured by using human PeripheralBlood Mononuclear Cells (hereinafter referred to as “PBMC”) as aneffector in the presence of the antibody. First, peripheral blood wascollected from the healthy human donor and mixed with anticoagulant. Theobtained blood was loaded onto Ficoll-Plaque Plus (manufactured by GEHealthcare), and centrifuged at 2000 rpm for 20 min using a large scalecentrifuge (CF9RX, manufactured by Hitachi Koki Co., Ltd.) so as not todisturb the boundary.

The middle fraction containing cells were collected and washed with PBS,and then centrifuged at 900 rpm for 20 min to remove the platelets. Thusobtained PBMC was used for the measurement. Next, the Daudi cell used asa target cell and the PBMC were mixed at the ratio ofeffector/target=50.

Each of the recombinant anti-human monoclonal defucose antibodies(antibody 512, antibody 644, antibody 4545 and antibody 4177) preparedin Example 13 and Example 14, and anti-DNP antibody as a human IgG1control was suspended in the medium to give a final concentration of 1μg/ml and a total volume of 100 μl. After mixing, the solution wasincubated at 37° C. under 5% CO₂ for 4 hr. The lysis ratio of the targetcell was evaluated based on the amount of LDH released into the medium.The LDH activity and lysis ratio were calculated using CytoTox 96Non-Radioactive Cytotoxicity Assay (manufactured by Promega Corp.) inaccordance with the instructions attached thereto. As statisticalanalysis, standard Student's t-test was used wherein a sample having arisk rate (p) of less than 0.05 was considered as one which wassignificantly different.

As the result that the ADCC activity on Daudi cells was measured, it wasfound that antibody 4545 and antibody 4177 exhibited the significantincrease in the lysis ratio of the target cell as compared to anti-DNPantibody. This result suggests that anti-TIM-3 mouse antibody 8213 andanti-TIM-3 antibody which competes with anti-TIM-3 mouse antibody 8213have high ADCC activity.

Similarly, the ADCC activity was measured by using recombinantanti-TIM-3 human monoclonal defucose antibodies (antibody 4545 andantibody 4177) prepared in Example 13, commercially available anti-TIM-3monoclonal antibody 344823, and anti-DNP antibody as a negative control.

First, peripheral blood was collected from the healthy human donor andmixed with anticoagulant. The obtained blood was then loaded ontoFicoll-Plaque Plus (manufactured by GE Healthcare), and centrifuged at2000 rpm for 20 min using a large scale centrifuge (CF9RX, manufacturedby Hitachi Koki Co., Ltd.) so as not to disturb the boundary. The middlefraction containing cells were collected and washed with PBS, and thencentrifuged at 900 rpm for 20 min to remove the platelets. Thus obtainedPBMC fraction was used for the measurement of t the ADCC activity.

Next, the Daudi cell used as a target cell and the PBMC were mixed atthe ratio of effector/target=25. Each of the antibody 4545, antibody4177, antibody 344823, anti-DNP antibody and PBS were suspended in themedium to give a final concentration of 1 μg/ml and a total volume of100 μl. After mixing, the solution was incubated at 37° C. under 5% CO₂for 4 hr. The lysis ratio of the target cell was evaluated based onusing the amount of LDH released into the medium.

The LDH activity and the lysis ratio were calculated using CytoTox 96Non-Radioactive Cytotoxicity Assay (manufactured by Promega Corp.) inaccordance with the instructions attached thereto. As statisticalanalysis, standard Student's t-test was used wherein a sample having arisk rate (p) of less than 0.05 was considered as one which wassignificantly different. As the result of the ADCC activity on Daudicells was measured, it was found that antibody 4545 and antibody 4177exhibited the significant increase in the lysis ratio of the target cellas compared to anti-DNP antibody. On the other hand, antibody 344823exhibited no significant increase in the lysis ratio of the target cellas compared to anti-DNP antibody and PBS.

Example 18 Calculating the Bound/Dissociation Constant Using Anti-TIM-3Antibody

The bound/dissociation constant was analyzed using surface plasmonresonance based biosensor (Biacore, manufactured by GE Healthcare). Inbrief, an anti-human antibody or an anti-mouse antibody was immobilizedon the CM5 sensor chips. Next, the anti-TIM-3 human or mouse antibodywere made to flow to bind the chip, and then soluble extracellular humanTIM-3 prepared in Example 4 was made to flow to examine thebound/association constant of TIM-3 to the anti-TIM-3 antibody.Throughout the experimental process, basically the experimental methodprovided by GE Healthcare for calculating bound/dissociation constantwas used.

Specifically, CM5 was used as the sensor chip (Research grade,manufactured by GE Healthcare). First, CM5 chip was activated by flowingthrough a mixed solution of equal parts of 400 mmol/L EDC(N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrocholoride) and 100mmol/L NHS(N-hyderoxysuccinimide). Next, the anti-human antibodyattached to the Human Antibody Capture Kit (manufactured by GEHealthcare) was diluted with the solution attached to the kit and theobtained solution was made to flow so that the required amount of theantibody to an antibody for a human antibody was immobilized on the CM5chip.

For the mouse antibody, the antibody against mouse antibody wasimmobilized on the CM5 surface by diluting in the solution, which wasprovided in the Mouse Antibody Capture Kit (manufactured by GEHealthcare antibody). The activated chip surface was blocked by thepassing through 1 mol/L for ethanolamide hydrochloride and inactivated.

Next, one kind of the anti-TIM-3 antibodies was diluted with HBS-EPbuffer (manufactured by GE Healthcare) per one flow cell and passedthrough so as to bind the antibody for the human antibody or antibodyfor the mouse antibody immobilized on the chip surface. Then solubleextracellular human TIM-3 was flown through. To dissociate the boundanti-TIM-3 antibody from the soluble extracellular human TIM-3, eitherthe total volume of 3 mol/L of MgCl₂ which was attached to the HumanAntibody Capture Kit or Glycine-HCl (pH 1.7) which was attached to theMouse Antibody Capture Kit were flown through the cell.

The above described procedures were considered as one step, and thesimilar procedure was repeated using different concentrations of thesoluble extracellular human TIM-3, to collect data (sensorgram) forcalculating the bound/association constant. The concentration of solubleextracellular human TIM-3 used as an analyte was calculated by measuringthe absorbance at 280 nm and then converting the value of 1.4 OD to 1mg/mL.

The molecular weight of the soluble extracellular human TIM-3 wascalculated as 42.8 kDa based on the mobility on electrophoresis. Thedata was analyzed using Biaevaluation soft-ware (manufactured by GEHealthcare) according to the Biaevaluation Software Handbook.Specifically, the simultaneous Kinetics analysis was performed and theassociation rate constant (Ka) and dissociation rate constant (Kd) wascalculated, by basically adopting a 1:1 Langmuir Binding reaction modelwas used for fitting. Then the value of the dissociation constant(K_(D)) was calculated from Kd/Ka.

Most of the anti-human TIM-3 human monoclonal antibody had the K_(D)value of the order of 10⁻⁹ mol/L. In this aspect, there was nocorrelation between ADCC activity and binding affinity targeting forTIM-3.

When the anti-human TIM-3 monoclonal antibody was monitored (e.g.,monitored the dissociation state for 30 min), there was no dissociationdetected with the antigen soluble extracellular human TIM-3 as anantigen. As a result, it was found that it was possible to produce amonoclonal antibody having a very high affinity for human TIM-3.

Example 19 Classification of Epitopes Using Competition Test-2

The epitopes of anti-TIM-3 antibodies, i.e., antibody 512, antibody 644,antibody 4545, antibody 8213, antibody 344823, antibody F38-2E2 andanti-DNP antibody as a control were analyzed by a competitive assay. Inbrief, the unlabeled target antibody was allowed to interact with theTIM-3 expression cell prepared in Example 1, and whether or not otherfluorescence labeled anti-TIM-3 antibody was further bound to TIM-3expression cells were evaluated by flow cytometry method.

Human TIM-3 expressing EoL-1 cells were washed with staining medium andallowed to react with purified test monoclonal antibodies [antibody 512,antibody 644, antibody 4545, antibody 8213, antibody 344823(manufactured by R&D Systems), antibody F38-2E2 (manufactured byImgenex)] (4° C., 30 min). The final concentration was 10 μg/ml. Next,the Alexa-647 or APC labeled anti-TIM-3 antibodies [antibody 344823(manufactured by R&D Systems) and antibody F38-2E2 (manufactured byeBiosciences)] were added and were allowed to react (4° C., 30 min).After washing with staining medium, 7-ADD (manufactured by BDBiosciences) was added before analyzing with FACSCalibur (manufacturedby BD Biosciences).

The results are shown in Table 1. As shown in Table 1, the case thedegree of the binding to the TIM-3 expression cell to which theunlabeled anti-TIM-3 antibody was bound was similar to that of anegative control were indicated as “+”, the case where the degree to thebinding to the TIM-3 expression cell to which the unlabeled anti-TIM-3antibody was bound was found but was lower than that of a negativecontrol was indicated as “+/−” and the case where the binding to theTIM-3 expression cell to which the unlabeled anti-TIM-3 antibody wasbound was not detected, it was indicated as “−”.

TABLE 1 Unlabeled antibody F38- DNP 512 644 4545 8213 344823 2E2 Alexa-DNP − − − − − − − 647 or 512 + − + + + + + APC 644 + + +/− + + +/− +/−labeled 4545 + + + − − + + antibody 8213 + + + − − + + 344823 + + − + +− +/− F38- + + − + + − − 2E2

As shown in Table 1, since Alexa-647 labeled antibody 512 was found tobind to the TIM-3 expressing cell to which the other unlabeledanti-TIM-3 antibodies were bound, this antibody was suggested torecognize an independent epitope with which antibody 644, antibody 4545,antibody 8213, antibody 344823 and antibody F38-2E2 did not compete.

Since it was found that Alexa-647 labeled antibody 644 did not bind orreduced in binding to the TIM-3 expressing cell to which the unlabeledanti-TIM-3 antibody 344823 or F38-2E2 was bound, it was indicated thatthis antibody competed with antibody 344823 or F38-E2E in therecognition of the epitope close to each other.

Since it was found that Alexa-647 labeled antibody 4545 did not bind tothe TIM-3 expressing cell to which the unlabeled anti-TIM-3 antibody8213 was bound, it was indicated that this antibody competed withantibody 8213 in the recognition of the epitope close to each other.

Since it was found that Alexa-647 labeled antibody 8213 did not bind tothe TIM-3 expressing cell to which the unlabeled anti-TIM-3 antibody4545 was bound, it was indicated that this antibody competed withantibody 4545 in the recognition of the epitope close to each other.

Since it was found that APC labeled antibody 344823 did not bind to theTIM-3 expressing cell to which the unlabeled anti-TIM-3 antibody 644 orF38-2E2 was bound, it was indicated that this antibody competed withantibody 644 or F38-E2E antibody in the recognition of the epitope closeto each other.

Since it was found that APC labeled Antibody F38-2E2 did not bind to theTIM-3 expressing cell to which the unlabeled anti-TIM-3 antibody 644 or344823 was bound, it was indicated that this antibody competed withantibody 644 or antibody 344823 in the recognition of the epitope closeto each other.

Example 20 Molecular Cloning of Mouse TIM-3 cDNA

The cDNA of mouse TIM-3 was prepared in the same manner as described inExample 1. Total RNA was extracted from Balb/c mouse derived bone marrowcells using High Pure RNA isolation Kit (manufactured by Roche).Template cDNA was synthesized using ThermoScript RT-PCR system(manufactured by Invitrogen). For primers, mTim-3 Fw3 (SEQ ID NO: 70)and mTim-3 Re3 (SEQ ID NO: 71) were used. The cDNA was inserted intopGEM-T Easy Vector (manufactured by Promega Corp.), and the sequence wasanalyzed. The clone having identical nucleotide sequence with the codingregion of GenBank accession number AF450241 was selected.

PCR was carried out using the selected clone as the template andinserting the DNA into NotI site of the pEF6/Myc-HisC vector(manufactured by Invitrogen) (mTim-3/pEF6 Myc_HisC). For PCR primers,mTim-3 Fw4NotI (SEQ ID NO: 72) and mTim-3 Re4 NotI (SEQ ID NO: 73) wereused.

Example 21 Construction of hTim-3/pEF6 Myc_HisC

The plasmid DNA hTIM-3/pMCs-IG prepared in Example 1 and pEF6 Myc_HisCwere both digested with NotI to construct human TIM-3 expressing pEF6Myc_HisC vector (hTim-3/pEF6 Myc_HisC). DNA sequence analysis confirmedthat the vector sequence matched the coding region of the GenBankaccession number NM_032782.

Example 22 Cross-Reactivity of Anti-Human TIM-3 Antibody to Anti-MouseTIM-3 Antibody

Each of the plasmid DNAs, mTim-3/pEF6 Myc_HisC and hTim-3/pEF6 Myc_HisCprepared in Example 20 and Example 21, and an empty vector control wasintroduced into HEK293F cell in the same manner as Example 4. Two dayslater, in the same manner as Example 15, the binding activity ofAlexa-647 labeled antibodies (antibody 512, antibody 644, antibody 4545and antibody 8213), commercially available APC labeled anti-human TIM-3antibodies (antibody 344823 and antibody F38-2E2) and commerciallyavailable PE labeled anti-mouse TIM-3 antibody (RMT3-23 antibody,manufactured by BioLegend Inc.) to human TIM-3 and mouse TIM-3 wasevaluated using flow cytometry analysis.

As a result, antibody 512, antibody 644, antibody 4545, antibody 8213,antibody 344823 and antibody F38-2E2 exclusively interacted with humanTIM-3 expressing 293F cells. However, RMT3-23 antibody interactedexclusively with mouse TIM-3 expressing 293F cells. Therefore, it wasfound that antibody 512, antibody 644, antibody 4545, antibody 8213,antibody 344823 and antibody F38-2E2 did not cross-react with mouseTIM-3.

Example 23 Construction of Expression System for TIM-3 Chimeric Proteinwith IgV Domain Substituted for Mouse TIM-3

The objective sequence was amplified by the PCR using hTim-3/pEF6Myc_HisC plasmid DNA as a template, hTIM3+mIgV_vecR1 primer (SEQ ID NO:74) and hTIM3+mIgV_vecF1 primer (SEQ ID NO: 75), and PrimeSTAR GXL DNAPolymerase (manufactured by Takara Bio Inc.). The objective sequence wasamplified by PCR using mTim-3/pEF6 Myc_HisC plasmid DNA as a template,hTIM3+mIgV_insF1 primer (SEQ ID NO: 76) and hTIM3+mIgV_insR1 primer (SEQID NO: 77), and PrimeSTAR GXL DNA Polymerase (manufactured by Takara BioInc.).

The two PCR products obtained were ligated using GENEART seamlesscloning and assembly kit (manufactured by Invitrogen). The sequence fromthe transformant was analyzed and it was found that the PCR products hadthe objective sequences (IgV chimeraTIM-3/pEF6 Myc_HisC, SEQ ID NO: 78).

Example 24 Construction of Expression System for TIM-3 Chimeric Proteinwith Mucin Domain Substituted for Mouse TIM-3

The objective sequence was amplified by PCR using hTim-3/pEF6 Myc_HisCplasmid DNA as a template, hTIM3+mMucin_vecR2 primer (SEQ ID NO: 80) andhTIM3+mMucin_vecF2 primer (SEQ ID NO: 81), and PrimeSTAR GXL DNAPolymerase (manufactured by Takara Bio Inc.). The objective sequence wasamplified by PCR using mTim-3/pEF6 Myc_HisC plasmid DNA as a template,hTIM3+mMuicn_insF2 primer (SEQ ID NO: 82) and hTIM3+mMucin_insR2 primer(SEQ ID NO: 83), and PrimeSTAR GXL DNA Polymerase (manufactured byTakara Bio Inc.).

The two PCR products obtained were ligated using GENEART seamlesscloning and assembly kit (manufactured by Invitrogen). The sequence fromthe transformant was analyzed and it was found that the PCR products hadthe objective sequences (Mucin chimeraTIM-3/pEF6 Myc_HisC, SEQ ID NO:84).

Example 25 Binding Assay of Anti-TIM-3 Antibody to TIM-3 ChimericProtein in which IgV Domain was Substituted with Mouse TIM-3 and TIM-3Chimeric Protein in which Mucin Domain was Substituted with Mouse TIM-3

Each of the plasmid DNAs prepared in Example 23 and Example 24, IgVchimeraTIM-3/pEF6 Myc_HisC, Mucin chimeraTIM-3/pEF6 Myc_HisC,hTim-3/pEF6 Myc_HisC, mTim-3/pEF6 Myc_HisC, and an empty vector controlwas introduced into HEK293F cell in the same manner as Example 4. Twodays later, in the same manner as Example 22, the binding activity ofanti-TIM-3 antibody was evaluated using flow cytometry.

As a result, antibody 512, antibody 644, antibody 4545, antibody 8213,antibody 4177, antibody 344823 and antibody F38-2E2 bound to the humanTIM-3 expressing cells and Mucin chimeraTIM-3 expressing cells only.However, RMT-23 antibody bound to the mouse TIM-3 expressing cells andthe IgV chimeraTIM-3 expressing cells only.

Therefore, as a result, it was found that all of the anti-human TIM-3antibodies tested (antibody 512, antibody 644, antibody 4545, antibody8213, antibody 344823 and antibody F38-2E2) and mouse TIM-3 antibody(antibody RMT3-23) recognized the IgV domain.

Example 26 Construction of TIM-3 Chimera 22-47/pEF6 Myc_HisC

A vector which expressed TIM-3 chimeric protein in which the amino acidof human TIM-3 (SEQ ID NO: 53) at position 22 to position 47 weresubstituted with the corresponding amino acid of mouse TIM-3(hereinafter referred to as “TIM-3 chimera 22-47”) was constructed. Byusing hTim-3/pEF6 Myc_HisC plasmid DNA prepared in Example 21 as atemplate, and hTIM3chimera22-47F1 primer (SEQ ID NO: 86) andhTIM3chimera22-47R1 primer (SEQ ID NO: 87), and PCR was carried outusing PrimeSTAR GXL DNA Polymerase (manufactured by Takara Bio Inc.).

Using the obtained PCR product as a template, the objective sequence wasamplified by PCR using hTIM3chimera22-47F2 primer (SEQ ID NO: 88) andhTIM3chimera22-47R2 primer (SEQ ID NO: 89) and using PrimeSTAR GXL DNAPolymerase (manufactured by Takara Bio Inc.).

Using the obtained PCR product as a template, the objective sequence wasamplified by PCR using hTIM3chimera22-47F3 primer (SEQ ID NO: 90) andhTIM3chimera22-47R3 primer (SEQ ID NO: 91), and using PrimeSTAR GXL DNAPolymerase (manufactured by Takara Bio Inc.).

After the obtained PCR product obtained from the third PCR was digestedwith DpnI (manufactured by New England Biolabs), it was subjected toagarose gel electrophoresis. The DNA was extracted and ligated usingGENEART seamless cloning and assembly kit (manufactured by Invitrogen).The sequence of the clone obtained from the transformant was analyzedand confirmed that the clone had the objective sequence (TIM-3 chimera22-47/pEF6 Myc_HisC, SEQ ID NO: 92).

Example 27 Construction of TIM-3 Chimera 57-66/pEF6 Myc_HisC

A vector which expressed TIM-3 chimeric protein in which the amino acidof human TIM-3 (SEQ ID NO: 53) at position 57 to position 66 weresubstituted with the corresponding amino acid of mouse TIM-3(hereinafter referred to as “TIM-3 chimera 57-66”) was constructed. PCRwas carried out using hTim-3/pEF6 Myc_HisC plasmid DNA prepared inExample 21 as a template, hTIM3chimera57-66F primer (SEQ ID NO: 94) andhTIM3chimera57-66R primer (SEQ ID NO: 95) phosphorylated by T4Polynucleotide Kinase (manufactured by New England Biolabs) andPrimeSTAR GXL DNA Polymerase (manufactured by Takara Bio Inc.) toamplify the objective sequence.

After the obtained PCR product was digested with DpnI (manufactured byNew England Biolabs), it was subjected to the agarose gelelectrophoresis. The DNA was extracted and ligated with LigaFast™ RapidDNA Ligation System (manufactured by Promega Corp.). The sequence of theclone obtained from the transformant was analyzed and confirmed that itwas the objective sequence (TIM-3 chimera 57-66/pEF6 Myc_HisC, SEQ IDNO: 96).

Example 28 Construction of TIM-3 Chimera 67-105/pEF6 Myc_HisC

A vector which expressed TIM-3 chimeric protein in which the amino acidof human TIM-3 (SEQ ID NO: 53) at position 67 to position 105 weresubstituted with the corresponding amino acid of mouse TIM-3(hereinafter referred to as “TIM-3 chimera 67-105”) was constructed. PCRwas carried out using hTim-3/pEF6 Myc_HisC plasmid DNA as a template,hTIM3chimera67-105F primer (SEQ ID NO: 98) and hTIM3chimera67-105Rprimer (SEQ ID NO: 99) and using PrimeSTAR GXL DNA Polymerase(manufactured by Takara Bio Inc.) to amplify the objective sequence.

PCR was carried out using mTim-3/pEF6 Myc_HisC plasmid DNA as atemplate, mTIM3chimera67-105F primer (SEQ ID NO: 100) andmTIM3chimera67-105R primer (SEQ ID NO: 101) and using PrimeSTAR GXL DNAPolymerase (manufactured by Takara Bio Inc.). The two PCR products wereligated by GENEART seamless cloning and assembly kit (manufactured byInvitorgen). The sequence of the clone obtained from the transformantwas analyzed and confirmed that it was the objective sequence (TIM-3chimera 67-105/pEF6 Myc_HisC, SEQ ID NO: 102).

Example 29 Construction of TIM-3 Chimera 74-81/pEF6 Myc_HisC

A vector which expressed TIM-3 chimeric protein in which the amino acidof human TIM-3 (SEQ ID NO: 53) at position 74 to position 81 weresubstituted with the corresponding amino acid of mouse TIM-3(hereinafter referred to as “TIM-3 chimera 74-81”) was constructed. PCRwas carried out using hTim-3/pEF6 Myc_HisC plasmid DNA prepared inExample 21 as a template, hTIM3chimera74-81F primer (SEQ ID NO: 104) andhTIM3chimera74-81R primer (SEQ ID NO: 105) and using PrimeSTAR GXL DNAPolymerase (manufactured by Takara Bio Inc.).

After the PCR product was digested with DpnI (manufactured by NewEngland Biolabs), it was subjected to agarose gel electrophoresis. TheDNA was extracted and ligated with GENEART seamless cloning and assemblykit (manufactured by Invitrogen). The sequence of the clone obtainedfrom the transformant was analyzed and confirmed that it was theobjective sequence (TIM-3 chimera 74-81/pEF6 Myc_HisC, SEQ ID NO: 106).

Example 30 Construction of TIM-3 Chimera 88-96/pEF6 Myc_HisC

A vector which expressed TIM-3 chimeric protein in which the amino acidof human TIM-3 (SEQ ID NO: 53) at position 88 to position 96 weresubstituted with the corresponding amino acid of mouse TIM-3(hereinafter referred to as “TIM-3 chimera 88-96”) was constructed. ThePCR was carried out by preparing a reaction solution containinghTim-3/pEF6 Myc_HisC plasmid DNA prepared in Example 21 as a template,hTIM3chimera88-96F primer (SEQ ID NO: 108) and hTIM3chimera88-96R primer(SEQ ID NO: 109) and using PrimeSTAR GXL DNA Polymerase (manufactured byTakara Bio Inc.).

After the obtained PCR product was digested with DpnI (manufactured byNew England Biolabs), it was subjected to agarose gel electrophoresis.The DNA was extracted and ligated with GENEART seamless cloning andassembly kit (manufactured by Invitrogen). The sequence of the cloneobtained from the transformant was analyzed and confirmed that it wasthe objective sequence (TIM-3 chimera 88-96/pEF6 Myc_HisC, SEQ ID NO:110).

Example 31 Construction of TIM-3 Chimera 96-105/pEF6 Myc_HisC

A vector which expressed TIM-3 chimeric protein in which the amino acidof human TIM-3 (SEQ ID NO: 53) at position 96 to position 105 weresubstituted with the corresponding amino acid of mouse TIM-3(hereinafter referred to as “TIM-3 chimera 96-105”) was constructed. ThePCR was carried out by preparing a reaction solution containinghTim-3/pEF6 Myc_HisC plasmid DNA prepared in Example 21 as a template,hTIM3chimera96-105F primer (SEQ ID NO: 112) and hTIM3chimera96-105Rprimer (SEQ ID NO: 113) and using PrimeSTAR GXL DNA Polymerase(manufactured by Takara Bio Inc.).

After the obtained PCR product was digested with DpnI (manufactured byNew England Biolabs), it was subjected to agarose gel electrophoresis.The DNA was extracted and ligated with GENEART seamless cloning andassembly kit (manufactured by Invitrogen). The sequence of the cloneobtained from the transformant was analyzed and confirmed that it wasthe objective sequence (TIM-3 chimera 96-105/pEF6 Myc_HisC, SEQ ID NO:114).

Example 32 Binding Assay of Anti-TIM-3 Antibody to TIM-3 ChimericProtein in which Part of the IgV Domain was Substituted with Mouse TIM-3

Each of various TIM-3 vectors prepared in Examples 26 to 31 and an emptyvector as a control were introduced into HEK293F in the same manner asExample 4.

The introduced vectors and the expressed TIM-3 chimeric proteins thereofwere as follows:

(1) TIM-3 chimera 22-47: TIM-3 chimeric protein in which the amino acidresidues at position 22 to position 47 of human TIM-3 (SEQ ID NO: 53)were substituted with the corresponding amino acids of mouse TIM-3;

(2) TIM-3 chimera 57-66: TIM-3 chimeric protein in which the amino acidresidues at position 57 to position 66 of human TIM-3 (SEQ ID NO: 53)were substituted with the corresponding amino acids of mouse TIM-3;

(3) TIM-3 chimera 67-105: TIM-3 chimeric protein in which the amino acidresidues at position 67 to position 105 of human TIM-3 (SEQ ID NO: 53)were substituted with the corresponding amino acids of mouse TIM-3;

(4) TIM-3 chimera 74-81: TIM-3 chimeric protein in which the amino acidresidues at position 74 to position 81 of human TIM-3 (SEQ ID NO: 53)were substituted with the corresponding amino acids of mouse TIM-3;

(5) TIM-3 chimera 88-96: TIM-3 chimeric protein in which the amino acidresidues at position 88 to position 96 of human TIM-3 (SEQ ID NO: 53)were substituted with the corresponding amino acids of mouse TIM-3; and

(6) TIM-3 chimera 96-105: TIM-3 chimeric protein in which the amino acidresidues at position 96 to position 105 of human TIM-3 (SEQ ID NO: 53)were substituted with the corresponding amino acids of mouse TIM-3.

Two days after transfection, in the same manner as Example 22, thebinding activity of anti-TIM-3 antibody was evaluated using flowcytometry.

The results are shown in Table 2. As shown in Table 2, the case wherethe binding of the antibody to the transfected cell was detected wasindicated as “+”, the case where the binding of the antibody to thetransfected cell was detected but the degree of the binding was reducedwas indicated as “+/−” and the case where the binding of the antibody tothe transfected cell was not detected was indicated as “−”.

TABLE 2 Vector name 4545 8213 4177 512 644 344823 F38-2E2 TIM-3chimera + + +/− − + + + 22-47 TIM-3 chimera + + +/− + − − − 57-66 TIM-3chimera − − +/− + + + + 67-105 TIM-3 chimera − − + + + + + 74-81 TIM-3chimera + + + + + + + 88-96 TIM-3 chimera + + + + + + + 96-105

As shown in Table 2, antibody 4545 and antibody 8213 bound to the cellstransfected with TIM3-chimera 22-47, TIM3-chimera 57-66, TIM3-chimera88-96 or TIM3-chimera 96-105 transfected cells. However, theseantibodies did not bind to the cell into which TIM-3 chimera 67-105 wereintroduced.

The antibody 512 bound to the cells transfected with TIM3-chimera 57-66,TIM3-chimera 88-96, TIM3-chimera 96-105, TIM-3 chimera 67-105 or TIM-3chimera 74-81. However, antibody 512 did not bind to the cellstransfected with TIM-3 chimera 22-47.

The antibody 644, antibody 344823 and antibody F38-2E2 bound to thecells transfected with TIM3-chimera 22-47, TIM3-chimera 88-96,TIM3-chimera 96-105, TIM-3 chimera 67-105 or TIM-3 chimera 74-81.However, these antibodies did not bind to the cells transfected with toTIM-3 chimera 57-66.

The antibody 4177 bound to the cells transfected with TIM3-chimera88-96, TIM3-chimera 96-105 or TIM-3 chimera 74-81. Antibody 4177 weaklybound to TIM-3 the cells transfected with chimera 22-47, TIM-3 chimera57-66 or TIM-3 chimera 67-105.

Based on the above result and results from Example 16 and Example 19, itwas suggested that antibody 4545 and antibody 8213 bound to the aminoacid residues at position 67 to 87 of human TIM-3. Since antibody 4545and antibody 8213 did not bind to the cells transfected with TIM-3chimera 74-81, it was suggested that the amino acids required forbinding of antibody 4545 and antibody 8213 to human TIM-3 were includedin the amino acid at position 74 to 81.

In addition, it was suggested that antibody 512 bound to the amino acidsup to at position 47 of human TIM-3 and that antibody 644, antibody344823 and antibody F38-2E2 bound to the amino acid residues at position57 to 66 of human TIM-3.

Furthermore, it was suggested that antibody 4177 bound to the aminoacids other than at position 74 to 81 within the amino acids at position67 to 87 of human TIM-3 to which antibody 4545 and antibody 8213 bindalthough antibody 512, antibody 644, antibody 344823 and antibodyF38-2E2 did not bind.

Example 33 Preparation of Anti-Human TIM-3 Antibody 8213 HumanizedAntibody

(1) Design of Amino Acid Sequences of VH and VL of Antibody 8213Humanized Antibody

The amino acid sequence of VH of antibody 8213 humanized antibody wasdesigned in the following manner. The amino acid sequences of FRs in VHof human antibody which is suitable for the grafting of the amino acidsequences (SEQ ID NOs: 21 to 23) of CDRs1 to 3 of antibody 8213 wereselected as follows.

Kabat et al., have classified the VH of conventionally known varioushuman antibodies into three subgroups (HSG I to III) based on thehomology of their amino acid sequences and reported the consensussequences for each of the subgroups [Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991)].Therefore, the homology search of the amino acid consensus sequences ofFR of VH subgroups I to III of human antibodies with the amino sequenceof FR of VH of antibody 8213 was conducted.

As a result of the homology search, the homologies of HSGI, HSGII, andHSGIII were 77.0%, 55.2%, and 58.6%, respectively. Therefore, the aminoacid sequence of FR of the VH region of antibody 8213 had the highesthomology to the subgroup I.

Based on the above results, the amino acid sequence of CDR of the VH ofantibody 8213 (SEQ ID NOs: 21 to 23) was grafted to an appropriateposition of the FR amino acid sequence of the consensus sequence ofsubgroup I of human antibody VH. In this manner, the 8213 antibody HV0represented by SEQ ID NO: 67, i.e., the amino acid sequence of VH of ananti-human TIM-3 antibody 8213 humanized antibody was designed.

Next, the amino acid sequence of VL of antibody 8213 humanized antibodywas designed in the following manner. The amino acid sequences of FR inVL of human antibody which were suitable for the grafting of the aminoacid sequences (SEQ ID NOs: 24 to 26) of CDRs1 to 3 in VL of antibody8213 were selected as follows.

Kabat et al., have classified the VL of various known human antibodiesinto four subgroups (HSG I to IV) based on the homology of their aminoacid sequences and reported the consensus sequences for each of thesubgroups [Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)]. Therefore, the homology search amongthe FR amino acid consensus sequences of VL subgroup I to IV of humanantibodies were searched for sequence homology with FR amino sequence ofVL of antibody 8213.

As a result of the homology search, the homologies of HSGI, HSGII,HSGIII and HSGIV were 76.3%, 61.3%, 61.3%, and 68.8%, respectively.Therefore, the amino acid sequence of FR of the VL region of antibody8213 had the highest homology to the subgroup I.

Based on the above results, each of the amino acid sequences of CDRs ofthe VL of antibody 8213 (SEQ ID NOs: 24 to 26) was grafted into anappropriate position in the amino acid sequence of FR of the consensussequence of subgroup I of VL of an human antibody. In this manner, the8213 antibody LV0 represented by SEQ ID NO: 69, i.e., the amino acidsequence of VL of an anti-human TIM-3 antibody 8213 humanized antibody,was designed.

The amino acid sequence of 8213 antibody HV0 which was VH of antibody8213 humanized antibody; and the amino acid sequence of 8213 antibodyLV0 which was VL of antibody 8213 humanized antibody designed in theabove were the sequences in which only the amino acid sequences of CDRsof the mouse monoclonal antibody 8213 were grafted into the amino acidsequence of the selected FR of human antibody.

However, in general, it is known that a humanized antibody preparedmerely by grafting CDRs of a mouse antibody to FRs of a human antibodyhas a lower binding activity. In order to avoid decreasing of thebinding activity, attempts have been made in the preparation of ahumanized antibody to raise the lowered binding activity by modifyingthe amino acid residues which were considered to have influence on thebinding activity among the amino acid sequences of FRs of a humanantibody which are different from a mouse antibody as well as graftingamino acid sequences of CDRs. Therefore, in Examples, the inventorsdecided to identify and modify the amino acid residues of FR which wereconsidered to have influence on the binding activity in the followingmanner.

First, three-dimensional structure of the above designed antibody Vregion (hereinafter referred to as “HV0LV0”) comprising the amino acidsequence of antibody 8213 HV0 which was VH of the antibody 8213humanized antibody and the amino acid sequence of antibody 8213 LV0which was VL of antibody 8213 humanized antibody was constructed using acomputer modeling technique. Discovery Studio (manufactured by AccelrysInc.) was used for preparation of the three-dimensional structure anddisplay of the three-dimensional structure. A computer model of thethree-dimensional structure of V region of antibody 8213 was constructedin the same manner.

Furthermore, amino acid residues which were different from those ofantibody 8213 in the amino acid sequence of FRs of VH and VL of HV0LV0were selected, prepared an amino acid sequence in which such amino acidresidues were substituted with the corresponding amino acid residues of8213 antibody and then a three-dimensional structure model wasconstructed in the same manner. The amino acids which were considered tohave influence on the binding activity were selected by comparing thethree-dimensional structures of the V regions of antibody 8213 andHV0LV0 and the modified product.

As a result, as amino acid residues of FR in HV0LV0 which wereconsidered to change the three-dimensional structure of the antigenbinding region and have influence on the binding activity, Lys atposition 12, Val at position 20, Arg at position 38, Ala at position 40,Met at position 48, Arg at position 67, Val at position 68, Ile atposition 70, Ala at position 72, Thr at position 74, Arg at position 98and Val at position 113 were selected in antibody 8213 HV0; and Leu atposition 11, Ala at position 13, Val at position 15, Tyr at position 36,Ala at position 43, Pro at position 44, Leu at position 46, Phe atposition 71 and Thr at position 85 were selected in antibody 8213 LV0.

Among the selected amino acid residues, at least one or more amino acidresidues was substituted with amino acid residues which were present atthe corresponding sites of antibody 8213 and VHs and VLs of thehumanized antibody comprising various modifications were constructed.

Specifically, as for VH, at least one modification was introduced amongthe amino acid modifications for substituting Lys at position 12 withVal, Val at position 20 with Leu, Arg at position 38 with Lys, Ala atposition 40 with Arg, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ile at position 70 with Leu, Alaat position 72 with Val, Thr at position 74 with Lys, Arg at position 98with Gly, or Val at position 113 with Leu in the amino acid sequencerepresented by SEQ ID NO: 67.

As for VL, at least one modification was introduced among the amino acidmodifications for substituting Leu at position 11 with Met, Ala atposition 13 with Val, Val at position 15 with Leu, Tyr at position 36with Leu, Ala at position 43with Ser, Pro at position 44 with Phe, Leuat position 46 with Gly, Phe at position 71 with Tyr and Thr at position85 with Asp in the amino acid sequence represented by SEQ ID NO: 69.

As the antibody V region of 8213 antibody-humanized antibody in which atleast one amino acid existing in FR in HV0LV0 was modified, HV0LV0,HV0LV2, HV0LV4, HV0LV5, HV0LV6, HV0LV7, HV0LV9, HV3LV0, HV3LV2, HV3LV4,HV3LV5, HV3LV6, HV3LV7, HV3LV9, HV4LV0, HV4LV2, HV4LV4, HV4LV5, HV4LV6,HV4LV7, HV4LV9, HV5LV0, HV5LV2, HV5LV4, HV5LV5, HV5LV6, HV5LV7, HV5LV9,HV6LV0, HV6LV2, HV6LV4, HV6LV5, HV6LV6, HV6LV7, HV6LV9, HV7LV0, HV7LV2,HV7LV4, HV7LV5, HV7LV6, HV7LV7, HV7LV9, HV8LV0, HV8LV2, HV8LV4, HV8LV5,HV8LV6, HV8LV7, HV8LV9, HV10LV0, HV10LV2, HV10LV4, HV10LV5, HV10LV6,HV10LV7, HV10LV9, HV12LV0, HV12LV2, HV12LV4, HV12LV5, HV12LV6, HV12LV7,and HV12LV9 were designed.

The amino acid sequences of each of the H chain variable regions, HV3,HV4, HV5, HV6, HV7, HV8, HV10 and HV12; and the amino acid sequence ofthe L chain variable region, LV2, LV4, LV5, LV6, LV7 and LV9 were shownin FIG. 1 and FIG. 2, respectively.

(2) Preparation of 8213 Antibody-Humanized Antibody

DNA encoding the amino acid sequence of the variable region of 8213antibody-humanized antibody was designed using codons used in the DNAs(SEQ ID NO: 27 and 29) encoding the amino acid sequence of VH of 8213antibody and VL of 8213 antibody, respectively. When the amino acidmodification was introduced, DNA was designed using a codon which wasused in a mammal cell with a high frequency.

Using these DNA sequences, vectors for expressing an antibody wereconstructed and humanized antibodies were expressed.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on U.S. provisional application No.61/353,836, filed on Jun. 11, 2010, the entire contents of which areincorporated hereinto by reference. All references cited herein areincorporated in their entirety.

FREE TEXT OF SEQUENCE LISTING

-   SEQ ID NO: 1: The amino acid sequence of Human 4545 H chain CDR1-   SEQ ID NO: 2: The amino acid sequence of Human 4545 H chain CDR2-   SEQ ID NO: 3: The amino acid sequence of Human 4545 H chain CDR3-   SEQ ID NO: 4: The amino acid sequence of Human 4545 L chain CDR1-   SEQ ID NO: 5: The amino acid sequence of Human 4545 L chain CDR2-   SEQ ID NO: 6: The amino acid sequence of Human 4545 L chain CDR3-   SEQ ID NO: 11: The amino acid sequence of Human 4177 H chain CDR1-   SEQ ID NO: 12: The amino acid sequence of Human 4177 H chain CDR2-   SEQ ID NO: 13: The amino acid sequence of Human 4177 H chain CDR3-   SEQ ID NO: 14: The amino acid sequence of Human 4177 L chain CDR1-   SEQ ID NO: 15: The amino acid sequence of Human 4177 L chain CDR2-   SEQ ID NO: 16: The amino acid sequence of Human 4177 L chain CDR3-   SEQ ID NO: 21: The amino acid sequence of Mouse 8213 H chain CDR1-   SEQ ID NO: 22: The amino acid sequence of Mouse 8213 H chain CDR2-   SEQ ID NO: 23: The amino acid sequence of Mouse 8213 H chain CDR3-   SEQ ID NO: 24: The amino acid sequence of Mouse 8213 L chain CDR1-   SEQ ID NO: 25: The amino acid sequence of Mouse 8213 L chain CDR2-   SEQ ID NO: 26: The amino acid sequence of Mouse 8213 L chain CDR3-   SEQ ID NO: 31: The nucleotide sequence of primer TIM-3 Fw2-   SEQ ID NO: 32: The nucleotide sequence of primer TIM-3 Re2-   SEQ ID NO: 33: The nucleotide sequence of primer pMCs-Fw-   SEQ ID NO: 34: The nucleotide sequence of primer TIM3ED-FcReXba-   SEQ ID NO: 35: The nucleotide sequence of primer T7-   SEQ ID NO: 36: The nucleotide sequence of primer hTIM-3 Fw1-   SEQ ID NO: 37: The nucleotide sequence of insert hTIM-3 ECD Fc    fusion-   SEQ ID NO: 38: The amino acid sequence of insert hTIM-3 ECD Fc    fusion-   SEQ ID NO: 39: The nucleotide sequence of primer TIM3ED-FLAG4aa-   SEQ ID NO: 40: The nucleotide sequence of primer C-FLAG-NotR2-   SEQ ID NO: 41: The nucleotide sequence of primer BGH-R-   SEQ ID NO: 42: The nucleotide sequence of insert hTIM-3 ECD-   SEQ ID NO: 43: The amino acid sequence of insert hTIM-3 ECD-   SEQ ID NO: 44: The nucleotide sequence of primer hh-6-   SEQ ID NO: 45: The nucleotide sequence of primer hh-3-   SEQ ID NO: 46: The nucleotide sequence of primer hk-2-   SEQ ID NO: 47: The nucleotide sequence of primer hk-6-   SEQ ID NO: 48: The nucleotide sequence of primer mH_Rv1-   SEQ ID NO: 49: The nucleotide sequence of primer mH_Rv2-   SEQ ID NO: 50: The nucleotide sequence of primer mK_Rv1-   SEQ ID NO: 51: The nucleotide sequence of primer mK_Rv2-   SEQ ID NO: 66: The nucleotide sequence of 8213 antibody HV0 variable    region-   SEQ ID NO: 67: The amino acid sequence of 8213 antibody HV0 variable    region-   SEQ ID NO: 68: The nucleotide sequence of 8213 antibody LV0 variable    region-   SEQ ID NO: 69: The amino acid sequence of 8213 antibody LV0 variable    region-   SEQ ID NO: 70: The nucleotide sequence of primer mTim-3 Fw3-   SEQ ID NO: 71: The nucleotide sequence of primer mTim-3 Re3-   SEQ ID NO: 72: The nucleotide sequence of primer mTim-3 Fw4NotI-   SEQ ID NO: 73: The nucleotide sequence of primer mTim-3 Re4 NotI-   SEQ ID NO: 74: The nucleotide sequence of primer hTIM3+mIgV_vecR1-   SEQ ID NO: 75: The nucleotide sequence of primer hTIM3+mIgV_vecF1-   SEQ ID NO: 76: The nucleotide sequence of primer hTIM3+mIgV_insF1-   SEQ ID NO: 77: The nucleotide sequence of primer hTIM3+mIgV_insR1-   SEQ ID NO: 78: The nucleotide sequence of insert IgV    chimeraTIM-3/pEF6 Myc_HisC-   SEQ ID NO: 79: The amino acid sequence of insert IgV    chimeraTIM-3/pEF6 Myc_HisC-   SEQ ID NO: 80: The nucleotide sequence of primer hTIM3+mMucin_vecR2-   SEQ ID NO: 81: The nucleotide sequence of primer hTIM3+mMucin_vecF2-   SEQ ID NO: 82: The nucleotide sequence of primer hTIM3+mMucin_insF2-   SEQ ID NO: 83: The nucleotide sequence of primer hTIM3+mMucin_insR2-   SEQ ID NO: 84: The nucleotide sequence of insert Mucin    chimeraTIM-3/pEF6 Myc_HisC-   SEQ ID NO: 85: The amino acid sequence of insert Mucin    chimeraTIM-3/pEF6 Myc_HisC-   SEQ ID NO: 86: The nucleotide sequence of primer hTIM3chimera22-47F1-   SEQ ID NO: 87: The nucleotide sequence of primer hTIM3chimera22-47R1-   SEQ ID NO: 88: The nucleotide sequence of primer hTIM3chimera22-47F2-   SEQ ID NO: 89: The nucleotide sequence of primer hTIM3chimera22-47R2-   SEQ ID NO: 90: The nucleotide sequence of primer hTIM3chimera22-47F3-   SEQ ID NO: 91: The nucleotide sequence of primer    hTIM3chimera22-47R3SEQ ID NO: 92: The nucleotide sequence of insert    TIM-3 chimera 22-47/pEF6 Myc_HisC-   SEQ ID NO: 93: The amino acid sequence of insert TIM-3 chimera    22-47/pEF6 Myc_HisC-   SEQ ID NO: 94: The nucleotide sequence of primer hTIM3chimera57-66F-   SEQ ID NO: 95: The nucleotide sequence of primer hTIM3chimera57-66R-   SEQ ID NO: 96: The nucleotide sequence of insert TIM-3 chimera    57-66/pEF6 Myc_HisC-   SEQ ID NO: 97: The amino acid sequence of insert TIM-3 chimera    57-66/pEF6 Myc_HisC-   SEQ ID NO: 98: The nucleotide sequence of primer hTIM3chimera67-105F-   SEQ ID NO: 99: The nucleotide sequence of primer hTIM3chimera67-105R-   SEQ ID NO: 100: The nucleotide sequence of primer    mTIM3chimera67-105F-   SEQ ID NO: 101: The nucleotide sequence of primer    mTIM3chimera67-105R-   SEQ ID NO: 102: The nucleotide sequence of insert TIM-3 chimera    67-105/pEF6 Myc_HisC-   SEQ ID NO: 103: The amino acid sequence of insert TIM-3 chimera    67-105/pEF6 Myc_HisC-   SEQ ID NO: 104: The nucleotide sequence of primer hTIM3chimera74-81F-   SEQ ID NO: 105: The nucleotide sequence of primer hTIM3chimera74-81R-   SEQ ID NO: 106: The nucleotide sequence of insert TIM-3 chimera    74-81/pEF6 Myc_HisC-   SEQ ID NO: 107: The amino acid sequence of insert TIM-3 chimera    74-81/pEF6 Myc_HisC-   SEQ ID NO: 108: The nucleotide sequence of primer hTIM3chimera88-96F-   SEQ ID NO: 109: The nucleotide sequence of primer hTIM3chimera88-96R-   SEQ ID NO: 110: The nucleotide sequence of insert TIM-3 chimera    88-96/pEF6 Myc_HisC-   SEQ ID NO: 111: The amino acid sequence of insert TIM-3 chimera    88-96/pEF6 Myc_HisC-   SEQ ID NO: 112: The nucleotide sequence of primer    hTIM3chimera96-105F-   SEQ ID NO: 113: The nucleotide sequence of primer    hTIM3chimera96-105R-   SEQ ID NO: 114: The nucleotide sequence of insert TIM-3 chimera    96-105/pEF6 Myc_HisC-   SEQ ID NO: 115: The amino acid sequence of insert TIM-3 chimera    96-105/pEF6 Myc_HisC

What is claimed is:
 1. A monoclonal antibody or an antigen-bindingfragment, which binds to an extracellular region of human T-cellimmunoglobulin and mucin domain containing molecule-3 (TIM-3) whilecompeting with one antibody selected from the following (a) to (c): (a)an antibody or an antigen-binding fragment thereof, each comprising aheavy chain (H chain) and a light chain (L chain), whereincomplementarity determining regions (CDRs) 1 to 3 of said H chaincomprises the amino acid sequences of SEQ ID NOs: 1 to 3, respectively,and CDRs 1 to 3 of said L chain comprises the amino acid sequences ofSEQ ID NOs: 4 to 6, respectively, (b) an antibody or an antigen-bindingfragment thereof, each comprising an H chain and L chain, wherein CDRs 1to 3 of said H chain comprises the amino acid sequences of SEQ ID NOs:11 to 13, respectively, and CDRs 1 to 3 of said L chain comprises theamino acid sequences of SEQ ID NOs: 14 to 16, respectively, and (c) anantibody or an antigen-binding fragment thereof, each comprising an Hchain and L chain, wherein CDRs 1 to 3 of said H chain comprises theamino acid sequences of SEQ ID NOs: 21 to 23, respectively, and CDRs 1to 3 of said L chain comprises the amino acid sequences of SEQ ID NOs:24 to 26, respectively, wherein the monoclonal antibody or theantigen-binding fragment thereof binds to the amino acid residues atpositions 67 to 105, the amino acid residues at positions 67 to 96 orthe amino acid residues at positions 67 to 87 in the amino acid sequenceof IgV domain of human T-cell immunoglobulin and mucin domain containingmolecule-3 (TIM-3) of SEQ ID NO:
 53. 2. The monoclonal antibody or theantigen-binding fragment thereof according to claim 1, which binds tothe same epitope as an epitope to which one antibody selected from theabove (a) to (c) binds.
 3. The monoclonal antibody or theantigen-binding fragment thereof according to claim 1, which is arecombinant antibody.
 4. The monoclonal antibody or the antigen-bindingfragment thereof according to claim 3, which is a recombinant antibodyselected from a human chimeric antibody, a humanized antibody and ahuman antibody.
 5. The antigen binding fragment according to claim 1,which is selected from the group consisting of a Fab, a Fab′, a F(ab′)2,a single chain antibody (scFv), a dimerized V region (diabody), adisulfide stabilized V region (dsFv) and a peptide comprising CDR.
 6. Aprocess for producing a monoclonal antibody or an antigen-bindingfragment thereof that binds to an extracellular region of human T-cellimmunoglobulin and mucin domain containing molecule-3 (TIM-3), saidprocess comprising culturing a transformant comprising a DNA encodingthe monoclonal antibody or the antigen binding fragment thereofaccording to claim 1 in a culture medium to form and accumulate themonoclonal antibody or the antigen binding fragment thereof, andrecovering the monoclonal antibody or the antigen-binding fragmentthereof from the culture.
 7. A method for immunologically detecting ormeasuring human T-cell immunoglobulin and mucin domain containingmolecule-3 (TIM-3), which comprises using the monoclonal antibody or theantigen-binding fragment thereof according to claim
 1. 8. A method fordiagnosing a disease relating to a human T-cell immunoglobulin and mucindomain containing molecule-3 (TIM-3) positive cell, which comprisesdetecting or measuring a human TIM-3 positive cell or human TIM-3, usingthe monoclonal antibody or the antigen-binding fragment thereofaccording to claim
 1. 9. A pharmaceutical composition, comprising themonoclonal antibody or the antigen-binding fragment thereof according toclaim 1 and a pharmaceutically acceptable carrier.
 10. A monoclonalantibody or an antigen-binding fragment, which binds to an extracellularregion of human T-cell immunoglobulin and mucin domain containingmolecule-3 (TIM-3) while competing with one antibody selected from thefollowing (a) and (b): (a) an antibody or an antigen-binding fragmentthereof, each comprising an H chain variable region (VH) and a L chainvariable region (VL), wherein said VH and VL comprises the amino acidsequences of SEQ ID NO: 116 and SEQ ID NO: 117, respectively, and (b) anantibody or an antigen-binding fragment thereof, each comprising VH andVL comprises the amino acid sequences of SEQ ID NO: 118 and SEQ ID NO:119, respectively.
 11. The monoclonal antibody or the antigen-bindingfragment thereof according to claim 10, which binds to the same epitopeas an epitope to which one antibody selected from the above (a) and (b)binds.
 12. The monoclonal antibody or the antigen-binding fragmentthereof according to claim 10, which is a recombinant antibody.
 13. Themonoclonal antibody or the antigen-binding fragment thereof according toclaim 12, which is a recombinant antibody selected from a human chimericantibody, a humanized antibody and a human antibody.
 14. The monoclonalantibody or the antigen-binding fragment thereof according to claim 10,which binds to the amino acid residues at positions 67 to 105, the aminoacid residues at positions 67 to 96 or the amino acid residues atpositions 67 to 87 in the amino acid sequence of IgV domain of humanT-cell immunoglobulin and mucin domain containing molecule-3 (TIM-3) ofSEQ ID NO:
 53. 15. The antigen binding fragment according to claim 10,which is selected from the group consisting of a Fab, a Fab′, a F(ab′)2,a single chain antibody (scFv), a dimerized V region (diabody), adisulfide stabilized V region (dsFv) and a peptide comprising CDR.
 16. Aprocess for producing a monoclonal antibody or an antigen-bindingfragment thereof that binds to an extracellular region of human T-cellimmunoglobulin and mucin domain containing molecule-3 (TIM-3), saidprocess comprising culturing a transformant comprising a DNA encodingthe monoclonal antibody or the antigen binding fragment thereofaccording to claim 1 in a culture medium to form and accumulate themonoclonal antibody or the antigen binding fragment thereof, andrecovering the monoclonal antibody or the antigen-binding fragmentthereof from the culture.
 17. A method for immunologically detecting ormeasuring human T-cell immunoglobulin and mucin domain containingmolecule-3 (TIM-3), which comprises using the monoclonal antibody or theantigen-binding fragment thereof according to claim
 10. 18. A method fordiagnosing a disease relating to a human T-cell immunoglobulin and mucindomain containing molecule-3 (TIM-3) positive cell, which comprisesdetecting or measuring a human TIM-3 positive cell or human TIM-3, usingthe monoclonal antibody or the antigen-binding fragment thereofaccording to claim
 10. 19. A pharmaceutical composition, comprising themonoclonal antibody or the antigen-binding fragment thereof according toclaim 10 and a pharmaceutically acceptable carrier.